WPRE mutant constructs, compositions, and methods thereof

ABSTRACT

The present invention provides a mutated woodchuck post-transcriptional regulatory element (WPRE). In particular, the present invention relates to a mutated WPRE sequence that can efficiently express nucleotides of interest in a retroviral vector system. The present invention also relates to methods of delivering and expressing nucleotides of interest to a target cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Non-Provisional patent application claims priority to U.S.Provisional Patent Application No. 62/988,202, filed 11 Mar. 2020. Thisapplication is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “3000011-017001_Sequence_Listing_ST25.txt” createdon 10 Mar. 2021, and 247,701 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND 1. Field

The present disclosure relates to a vector comprising a mutatedpost-transcriptional regulatory element. In particular, the presentinvention relates to mutated WPRE sequences that can efficiently expressnucleotides of interest in a retroviral vector system. The presentinvention also relates to methods of delivering and expressingnucleotides of interest in a target cell.

2. Background

Retroviral vectors, such as lentiviral vectors, have been proposed as adelivery system for, inter alia, the transfer of a nucleotide ofinterest to one or more sites of interest.

One shortcoming of retroviral vectors, whether based on retroviruses orlentiviruses, is their frequent inability to generate high levels ofgene expression, particularly in vivo. Many steps, both transcriptionaland post-transcriptional, are involved in regulating gene expression.Therefore, it is possible to enhance expression of transgenes deliveredby retroviral vectors through the addition of elements known topost-transcriptionally increase gene expression. An example is theinclusion of introns within the expression cassette (Choi, T. et al,(1991) Mol. Cell. Biol. 9:3070-3074). Many gene transfer experiments,performed both in vitro and in vivo, have demonstrated that the presenceof an intron can facilitate gene expression.

Other types of elements can also be used to stimulate heterologous geneexpression post-transcriptionally. These elements, unlike introns, areadvantageous in that they do not require splicing events. For instance,previous studies have suggested that the hepatitis B virus (HBV)post-transcriptional regulatory element (PRE) and an intron arefunctionally equivalent (Huang, Z. M. and Yen, T. S. (1995) Mol. Cell.Biol. 15: 3864-3869). Woodchuck hepatitis virus (WHV), a close relativeof HBV, also harbors a PRE (hereinafter referred to as WPRE; see U.S.Pat. Nos. 6,136,597 and 6,287,814). The WPRE has been shown to be moreactive than its HBV counterpart, correlating to the presence ofadditional cis-acting sequences not found in the HBV PRE. Insertion ofthe WPRE in lentiviral vectors resulted in significant stimulation ofexpression of reporter genes such as luciferase and green fluorescentprotein (GFP) in a variety of cells spanning different species(Zufferey, R. et al. (1999) J. Virol 73: 2886-2892). Stimulation wasirrespective of the cycling status of transduced cells.

The WPRE contains three cis-acting sequences important for its functionin enhancing expression levels. However, it also contains a fragment ofapproximately 180 base pairs (bp) comprising the 5′ end of the WHV Xprotein open reading frame, together with its associated promoter. Thefull-length X protein has been implicated in tumorigenesis (Flajolet, M.et al. (1998) J. Virol. 72: 6175-6180). Cis-activation of myc familyoncogenes due to the insertion of viral DNA into the host genome isknown to be a key mechanism of WHV-mediated carcinogenesis (Buendia, M.A. (1994) In C. Brechot (ed.), Primary liver cancer: etiological andprogression factors, p. 211-224: CRC Press, Boca Raton, Fla.; Fourel, G.(1994) In F. Tronche and M. Yaniv (ed.), Liver gene expression, p.297-343; R. G. Landes Company, Austin, Tex.). The tumorigenic potentialof the WHV X protein has raised concerns regarding inclusion of the WPREin retroviral vectors, in particular for in vivo applications.

Previous studies have suggested that mutation of the X protein openreading frame (ORF) within the WPRE reduces tumorigenic activity of theX protein, thereby improving its safety profile for inclusion inretroviral vectors (see, e.g., U.S. Pat. No. 7,419,829; Donello, J. E.et al. (1998) J. Virol. 72(6): 5085-5092; Schambach, A. et al. (2006)Gene Ther. 13: 641-645; Zanta-Boussif, M. A. et al. (2009) Gene Ther.16: 605-619; Ou L. et al. (2016) Mol. Gen. Metab. Rep. 8: 87-93).However, inconsistent effects on post-transcriptional stimulation ofheterologous gene expression have been seen in the various mutant WPREs.Generally, the greater the extent of mutations introduced into the WPRE,the less effective the mutant WPRE is in stimulatingpost-transcriptional heterologous gene expression.

Thus, there remains a need for safe and effective WPREs for use inretroviral vectors.

BRIEF SUMMARY

In an aspect, the present application relates to mutated WPRE sequencesfor use in, for example, retroviral vectors in which WHV X proteinexpression is attenuated or absent. In some embodiments, start codons ofany open reading frame (ORF) within WPRE are mutated, the WHV X proteinpromoter is deleted, and the WHV X protein OFR is deleted. In someembodiments, the WHV X protein promoter and WHV X protein start codon ismutated.

In some embodiments, the mutated WPRE sequence contains a mutation atone or more of the start codons corresponding to nucleotide positions106-108, 152-154, 245-247, 272-274, 283-285, 362-364, and 603-605 withinthe WT WPRE nucleotide sequence according to SEQ ID NO: 1. In someembodiments, the mutated WPRE sequence contains a mutation at one ormore of the start codons corresponding to nucleotide positions 70-72,108-110, 121-123, 138-140, 187-189, and 428-430 within the WT WPREnucleotide sequence according to SEQ ID NO: 2.

The start codon(s) may be mutated at one, two, or all three positionswithin the one or more start codons. If more than one start codon ismutated, each start codon mutation may be independent of the others. Inother words, each start codon mutated within the WPRE need not bemutated in an identical manner. In some embodiments, each of the one ormore start codon(s) is mutated at one position within the startcodon(s). For example, the first nucleotide of the start codon may bemutated from “A” to “C”, “G”, or “T”; or the second nucleotide of thestart codon may be mutated from “T” to “A”, “C”, or “G”, or the thirdnucleotide of the start codon may be mutated from “G” to “A”, “C”, or“T”. In some embodiments, one or more of the start codon(s) is mutatedfrom “ATG” to “TTG”. In some embodiments, each of the one or more startcodon(s) is mutated from “ATG” to “TTG”.

In some embodiments, the mutant WPRE sequence is selected from SEQ IDNO: 3 and SEQ ID NO: 4.

The present application also provides vectors, such as retroviral orlentiviral vectors, comprising the mutant WPREs of the invention. Suchvectors can be used in functional genomics, drug discovery, targetvalidation, protein production (e.g., therapeutic proteins, vaccines,monoclonal antibodies), gene therapy, and therapeutic treatments, suchas in gene delivery systems for adaptive cellular therapy.

In some aspects, lentiviral transduction vectors, and constructs fortheir manufacture, are provided which can be used to introduceexpressible nucleotide sequences of interest (NOI) into host cells. Alentiviral transduction vector is an enveloped virion particle thatcontains an expressible nucleotide sequence, and which is capable ofpenetrating a target host cell, thereby carrying the expressiblesequence into the cell. The enveloped particle is preferably pseudotypedwith an engineered or native viral envelope protein from another viralspecies, including non-lentiviruses, which alters the host range andinfectivity of the native lentivirus. As described in more detail below,the transduction vectors can be utilized in a wide range ofapplications, including, e.g., for protein production (including vaccineproduction), for gene therapy, to deliver therapeutic polypeptides, todeliver siRNA, ribozymes, anti-sense, and other functionalpolynucleotides, etc. Such transduction vectors have the ability tocarry single or multiple genes, and to include inhibitory sequences(e.g., RNAi or antisense).

In some aspects, the vector comprises more than one NOI. Such vectorscan be used, for example, to produce multimeric proteins in a host cell.In some aspects, the vector comprises a first nucleotide sequence S1encoding a protein Z1 and a second nucleotide sequence S2 encoding aprotein Z2, in which Z1 and Z2 form a dimer. The vector may furthercomprise a third nucleotide sequence S3 encoding a protein Y1, and afourth nucleotide sequence S4 encoding a protein Y2, in which Y1 and Y2form a second dimer.

In another aspect, the vector may further include a fifth nucleotidesequence S5 encoding a 2A peptide and a sixth nucleotide sequence S6encoding a linker peptide, wherein S5 and S6 are positioned between 51and S2, 51 and S3, 51 and S4, S2 and S3, S2 and S4, and/or S3 and S4.

In some aspects, the 2A peptide may be selected from P2A (SEQ ID NO: 6),T2A (SEQ ID NO: 7), E2A (SEQ ID NO: 8), or F2A (SEQ ID NO: 9).

In some aspects, the linker peptide is any peptide having a length of 3to 10 amino acid length. In some aspects, the linker peptide may be GSGor SGSG (SEQ ID NO: 5).

In another aspect, the vector may further include a seventh nucleotidesequence S7 encoding a furin peptide (SEQ ID NO: 10) positioned between51 and S2, 51 and S3, 51 and S4, S2 and S3, S2 and S4, and/or S3 and S4.

In another aspect, the vector may further include a promoter sequencethat controls the transcription of 51, S2, S3, S4, S5, S6 and/or S7,wherein the promoter sequence is selected from cytomegalovirus (CMV)promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein(MBP) promoter, glial fibrillary acidic protein (GFAP) promoter,modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer(MNDU3), Ubiqitin C promoter, EF-1 alpha promoter, or Murine Stem CellVirus (MSCV) promoter.

In some aspects, the first dimer Z1Z2 may be selected from SEQ ID NO: 13and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and26, 25 and 92, 91 and 92, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87and 88, or 89 and 90.

In some aspects, the second dimer Y1Y2 is set forth in SEQ ID NO: 11 and12.

In another aspect, the viral vector is selected from adenoviruses,poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses,retroviruses, lentiviruses, herpesviruses, paramyxoviruses, orpicornaviruses.

In another aspect, the vector is pseudotyped with an envelope protein ofa virus selected from the native feline endogenous virus (RD114), achimeric version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), achimeric version of GALV (GALV-TR), amphotropic murine leukemia virus(MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G),fowl plague virus (FPV), Ebola virus (EboV), baboon retroviral envelopeglycoprotein (BaEV), or lymphocytic choriomeningitis virus (LCMV).

In one aspect, the present disclosure relates to a method of preparing Tcells for immunotherapy including isolating T cells from a blood sampleof a human subject, activating the isolated T cells in the presence ofan aminobisphosphonate, transducing the activated T cells with thevector described herein, and expanding the transduced T cells.

In another aspect, the T cells may be isolated from a leukapheresishuman sample.

In another aspect, the aminobisphosphonate may be selected frompamidronic acid, alendronic acid, zoledronic acid, risedronic acid,ibandronic acid, incadronic acid, a salt thereof and/or a hydratethereof.

In another aspect, the T cells can be activated with OKT3 and anti-CD28.

In another aspect, the activating may be further in the presence ofhuman recombinant interleukin 2 (IL-2), human recombinant interleukin 15(IL-15), human recombinant interleukin 7 (IL-7).

In another aspect, the expanding may be in the presence of IL-2 andIL-15 or IL-15 and IL-7.

In another aspect, the T cells may be γδ T cells or αβ cells.

In another aspect, the first dimer Z1Z2 and the second dimer Y1Y2 areco-expressed on the surface of the expanded T cells.

In another aspect, the present disclosure relates to a population ofexpanded T cells prepared by the method of the above aspects.

In some aspects, the composition further includes an adjuvant.

In some aspects, the adjuvant is selected from one or more of anti-CD40antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib,bevacizumab, atezolizuma, interferon-alpha, interferon-beta, CpGoligonucleotides and derivatives, poly-(I:C) and derivatives, RNA,sildenafil, particulate formulations with poly(lactide co-glycolide)(PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13,IL-15, IL-21, and IL-23.

In one aspect, the present disclosure relates to a method of treating apatient who has cancer, comprising administering to the patient acomposition comprising the population of expanded T cells describedherein, in which the T cells kill cancer cells that present a peptide ina complex with an MHC molecule on the surface, wherein the peptide isselected from any of SEQ ID NO: 99-256, in which the cancer is selectedfrom the group consisting of non-small cell lung cancer, small cell lungcancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkelcell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer,colorectal cancer, urinary bladder cancer, kidney cancer, leukemia,ovarian cancer, esophageal cancer, brain cancer, gastric cancer, andprostate cancer.

In one aspect, the present disclosure relates to T cells describedherein or compositions comprising the population of expanded T cellsdescribed herein for use in the treatment of cancer, in which the canceris selected from the group consisting of non-small cell lung cancer,small cell lung cancer, melanoma, liver cancer, breast cancer, uterinecancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer,bile duct cancer, colorectal cancer, urinary bladder cancer, kidneycancer, leukemia, ovarian cancer, esophageal cancer, brain cancer,gastric cancer, and prostate cancer.

In a further aspect, the present disclosure refers to the use of T cellsdescribed herein or compositions comprising T cells described herein forthe manufacture of a medicament.

In a further aspect, the present disclosure refers to the use of T cellsdescribed herein or compositions comprising T cells described herein forthe manufacture of a medicament for the treatment of cancer, inparticular for the herein above-mentioned cancers.

In one aspect, the present disclosure relates to a method of elicitingan immune response in a patient who has cancer, comprising administeringto the patient a composition comprising the population of expanded Tcells described herein, in which the T cells kill cancer cells thatpresent a peptide in a complex with an MHC molecule on the surface,wherein the peptide is selected from any of SEQ ID NO: 99-256, and inwhich the cancer is selected from the group consisting of non-small celllung cancer, small cell lung cancer, melanoma, liver cancer, breastcancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer,gallbladder cancer, bile duct cancer, colorectal cancer, urinary bladdercancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer,brain cancer, gastric cancer, and prostate cancer.

In another aspect, the immune response comprises a cytotoxic T cellresponse.

Finally, the invention also provides kits comprising at least one vectorof the invention. In one embodiment, the kit comprises at least onevector of the invention, optionally packaging material, and optionally alabel or packaging insert contained within the packaging material.

In an aspect, the present disclosure relates to a method of preparing Tcells for immunotherapy, including isolating T cells from a blood sampleof a human subject, activating the isolated T cells in the presence of astatin, transducing the activated T cells with the vector of the presentdisclosure, in which the vector may be pseudotyped with any envelopeprotein described herein including vesicular stomatitis virus (VSV-G)and RD114TR, and expanding the transduced T cells.

In another aspect, the T cells may include CD4+ T cells, CD8+ T cells,γδ T cells, and/or natural killer T cells.

In another aspect, statin may be selected from atorvastatin,cerivastatin, dalvastatin, fluindostatin, fluvastatin, mevastatin,pravastatin, simvastatin, velostatin, and rosuvastatin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of a wild-type (WT) WPRE derived from thewoodchuck hepatitis virus genome provided in GenBank Accession No.J02440.1 (SEQ ID NO: 1), a WT WPRE derived from the woodchuck hepatitisB virus (strain WHV8) provided in GenBank Accession No. J04514.1 (SEQ IDNO: 2), a mutant WPRE in which the X protein promoter and start codon ismutated (SEQ ID NO: 4), and a mutant WPRE in which multiple start codonswithin the WPRE are mutated and both the X protein promoter and ORF aredeleted (SEQ ID NO: 3).

FIG. 2 shows an alignment of a wild-type (WT) WPRE derived from thewoodchuck hepatitis virus genome provided in GenBank Accession No.J02440.1 (SEQ ID NO: 1) and a mutant WPRE in which multiple start codonswithin the WPRE are mutated and both the X protein promoter and ORF aredeleted (SEQ ID NO: 3). The X protein promoter is underlined and the Xprotein start codon is italicized.

FIG. 3 shows a schematic diagram of vector constructs in accordance withsome embodiments of the present disclosure.

FIG. 4 shows exemplary lentiviral constructs in accordance with someembodiments of the present disclosure.

FIG. 5 shows HEK-293 T titers obtained following transduction withlentiviral constructs in accordance with some embodiments of the presentdisclosure. Variant A contains wild-type (WT) WPRE (positive control);variant B contains no WPRE (negative control); variant C contains amutant WPRE in which the X protein promoter and start codon are mutated(SEQ ID NO: 4); and variant D contains a mutant WPRE in which startcodons are mutated and both the X protein promoter and ORF are deleted(SEQ ID NO: 3).

FIG. 6 shows expression of TCRs on the surface of CD8+ cells six daysafter transduction with R4-B4 lentiviral constructs in accordance withsome embodiments of the present disclosure. Expression was detected bytetramer using lentiviral titration in two separate donors: Donor #1 inpanel A and donor #2 in panel B. Log viral dilution factor is presentedalong the X-axis. Variant A contains wild-type (WT) WPRE (positivecontrol); variant B contains no WPRE (negative control); variant Ccontains a mutant WPRE in which the X protein promoter and start codonare mutated (SEQ ID NO: 4); and variant D contains a mutant WPRE inwhich start codons are mutated and both the X protein promoter and ORFare deleted (SEQ ID NO: 3).

FIG. 7 shows expression of TCRs on the surface of CD8+ cells six daysafter transduction with R4-A1B4 lentiviral constructs in accordance withsome embodiments of the present disclosure. Expression was detected bytetramer using lentiviral titration in two separate donors: Donor #1 inpanel A and donor #2 in panel B. Log viral dilution factor is presentedalong the X-axis. Variant A contains wild-type (WT) WPRE (positivecontrol); variant B contains no WPRE (negative control); variant Ccontains a mutant WPRE in which the X protein promoter and start codonare mutated (SEQ ID NO: 4); and variant D contains a mutant WPRE inwhich start codons are mutated and both the X protein promoter and ORFare deleted (SEQ ID NO: 3).

FIG. 8 shows expression of TCRs on the surface of CD8+ cells (A) or CD4+cells (B) six days after transduction with R4-B4 lentiviral constructsin accordance with some embodiments of the present disclosure.Expression was detected by tetramer using lentiviral titration. Logviral dilution factor is presented along the X-axis. Variant A containswild-type (WT) WPRE (positive control); variant B contains no WPRE(negative control); variant C contains a mutant WPRE in which the Xprotein promoter and start codon are mutated (SEQ ID NO: 4); and variantD contains a mutant WPRE in which start codons are mutated and both theX protein promoter and ORF are deleted (SEQ ID NO: 3).

FIG. 9 shows expression of TCRs on the surface of CD8+ cells (A) or CD4+cells (B) six days after transduction with R4-A1B4 lentiviral constructsin accordance with some embodiments of the present disclosure.Expression was detected by tetramer using lentiviral titration. Logviral dilution factor is presented along the X-axis. Variant A containswild-type (WT) WPRE (positive control); variant B contains no WPRE(negative control); variant C contains a mutant WPRE in which the Xprotein promoter and start codon are mutated (SEQ ID NO: 4); and variantD contains a mutant WPRE in which start codons are mutated and both theX protein promoter and ORF are deleted (SEQ ID NO: 3).

FIG. 10 shows fold expansion is not affected by WPRE mutations. Cellviability was higher than 90% for all lentiviral constructs tested atoptimal MOI (data not shown). Description of the lentiviralabbreviations presented along the X-axis can be found in FIG. 4 .Briefly, the last letter in each construct abbreviation corresponds tothe WPRE used. Variant A contains wild-type (WT) WPRE (positivecontrol); variant B contains no WPRE (negative control); variant Ccontains a mutant WPRE in which the X protein promoter and start codonare mutated (SEQ ID NO: 4); and variant D contains a mutant WPRE inwhich start codons are mutated and both the X protein promoter and ORFare deleted (SEQ ID NO: 3).

FIG. 11 shows that WPRE mutants do not alter TCR tetramer expressionnormalized to vector copy number. Data presented is the mean of alldonors+/−standard deviation (SD). Panel A shows results for CD8+Tetramer+ only. Panel B shows results for total CD3+ Tetramer+.A=wild-type (WT) WPRE (positive control); B=no WPRE (negative control);C=mutant WPRE in which the X protein promoter and start codon aremutated (SEQ ID NO: 4); and D=mutant WPRE in which start codons aremutated and both the X protein promoter and ORF are deleted (SEQ ID NO:3).

FIG. 12 shows that WPRE mutants demonstrate comparable TCR tetramerexpression normalized to viral titer. Data presented is the mean of alldonors+/−standard deviation (SD). A=wild-type (WT) WPRE (positivecontrol); B=no WPRE (negative control); C=mutant WPRE in which the Xprotein promoter and start codon are mutated (SEQ ID NO: 4); andD=mutant WPRE in which start codons are mutated and both the X proteinpromoter and ORF are deleted (SEQ ID NO: 3).

FIG. 13 shows that WPRE mutants demonstrate comparable TCR tetramersurface expression as determined by flow cytometry. Panel A presentsCD4−CD8+/tetramer+ data. Panel B presents CD4+CD8−/tetramer+ data.A=wild-type (WT) WPRE (positive control); B=no WPRE (negative control);C=mutant WPRE in which the X protein promoter and start codon aremutated (SEQ ID NO: 4); and D=mutant WPRE in which start codons aremutated and both the X protein promoter and ORF are deleted (SEQ ID NO:3).

FIG. 14 shows cytokine production of CD4+ or CD8+ T cells in thepresence of target-positive tumor cells. Panel A presents interferon-γ(IFN-γ) production in CD8+ T cells. Panel B presents IFN-γ production inCD4+ T cells. Panel C presents tumor necrosis factor-α (TNF-α)production in C8+ T cells. Panel D presents TNF-α production in CD4+ Tcells. MCF7=negative; SW982=460 CpC. Description of the lentiviralabbreviations presented along the X-axis can be found in FIG. 4 .Briefly, the last letter in each construct abbreviation corresponds tothe WPRE used. Variant A contains wild-type (WT) WPRE (positivecontrol); variant B contains no WPRE (negative control); variant Ccontains a mutant WPRE in which the X protein promoter and start codonare mutated (SEQ ID NO: 4); and variant D contains a mutant WPRE inwhich start codons are mutated and both the X protein promoter and ORFare deleted (SEQ ID NO: 3).

FIG. 15 shows a γδ T cell manufacturing process according to oneembodiment of the present disclosure. γδ T cell manufacturing mayinclude collecting or obtaining white blood cells or PBMC, e.g.,leukapheresis product, depleting αβ T cells from PBMC or leukapheresisproduct, followed by activation, transduction, and expansion of γδ Tcells.

FIG. 16 shows a T cell manufacturing process according to anotherembodiment of the present disclosure. T cell manufacturing may includecollecting or obtaining white blood cells or PMBC, e.g., leukapheresisproduct followed by activation, transduction, and expansion of T cells.

FIG. 17 shows γδ T cell manufacturing process in accordance with oneembodiment of the present disclosure.

FIG. 18A shows the effect of WPRE on transgene expression in γδ T cellsin accordance with one embodiment of the present disclosure.

FIG. 18B shows the effect of WPRE on transgene expression in γδ T cellsin accordance with another embodiment of the present disclosure.

FIG. 19A shows the effect of WPRE on transgene expression in γδ T cellsin accordance with another embodiment of the present disclosure.

FIG. 19B shows the effect of WPRE on transgene expression in γδ T cellsin accordance with another embodiment of the present disclosure.

FIG. 20 shows the effect of WPRE on copy numbers of integrated transgenein γδ T cells in accordance with one embodiment of the presentdisclosure.

FIG. 21 shows the effect of WPRE on transgene expression/copy number ofintegrated transgene ratios in γδ T cells in accordance with oneembodiment of the present disclosure.

FIG. 22 shows the effect of WPRE on transgene expression/copy number ofintegrated transgene ratios in γδ T cells in accordance with anotherembodiment of the present disclosure.

FIG. 23 shows the effect of WPRE on transgene expression in γδ T cellsin accordance with another embodiment of the present disclosure.

FIG. 24 shows the effect of WPRE on transgene expression/copy number ofintegrated transgene ratios in γδ T cells in accordance with anotherembodiment of the present disclosure.

FIG. 25 shows the effect of WPRE on transgene expression/copy number ofintegrated transgene ratios in γδ T cells in accordance with anotherembodiment of the present disclosure.

DETAILED DESCRIPTION

Before the subject disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments of the disclosure described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the presentdisclosure will be established by the appended claims.

As used herein, the term “operably linked” means that the componentsdescribed are in a relationship permitting them to function in theirintended manner.

As used herein, the term “self-cleaving 2A peptide” refers to relativelyshort peptides (of the order of 20 amino acids long, depending on thevirus of origin) acting co-translationally, by preventing the formationof a normal peptide bond between the glycine and last proline, resultingin the ribosome skipping to the next codon, and the nascent peptidecleaving between the Gly and Pro. After cleavage, the short 2A peptideremains fused to the C-terminus of the ‘upstream’ protein, while theproline is added to the N-terminus of the ‘downstream’ protein.Self-cleaving 2A peptide may be selected from porcine teschovirus-1(P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A),foot-and-mouth disease virus (F2A), or any combination thereof (see,e.g., Kim et al., PLOS One 6:e18556, 2011, the content of whichincluding 2A nucleic acid and amino acid sequences are incorporatedherein by reference in their entireties). By adding the linker sequences(such as GSG or SGSG (SEQ ID NO: 5)) before the self-cleaving 2Asequence, this may enable efficient synthesis of biologically activeproteins, e.g., TCRs.

As used herein, the term “promoter” refers to a regulatory region of DNAgenerally located upstream (towards the 5′ region of the sense strand)of a gene that allows transcription of the gene. The promoter containsspecific DNA sequences and response elements that are recognized byproteins known as transcription factors. These factors bind to thepromoter sequences, recruiting RNA polymerase, the enzyme thatsynthesizes the RNA from the coding region of the gene. For example, thepromoter sequence used herein may be selected from cytomegalovirus (CMV)promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein(MBP) promoter, glial fibrillary acidic protein (GFAP) promoter,modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer(MNDU3), Ubiqitin C promoter, EF-1 alpha promoter, or Murine Stem CellVirus (MSCV) promoter.

As used herein, the term “cistron” refers to a section of the DNAmolecule that specifies the formation of one polypeptide chain, i.e.coding for one polypeptide chain. For example, “bi-cistron” refers totwo sections of the DNA molecule that specify the formation of twopolypeptide chains, i.e. coding for two polypeptide chains;“tri-cistron” refers to three sections of the DNA molecule that specifythe formation of three polypeptide chains, i.e. coding for threepolypeptide chains; etc.

As used herein, the term “multi-cistronic RNA” or “multi-cistronic mRNA”refers to an RNA that contains the genetic information to translate toseveral proteins. In contrast, a mono-cistronic RNA contains the geneticinformation to translate only a single protein. In the context of thepresent disclosure, the multi-cistronic RNA transcribed from thelentivirus may be translated into translated to two proteins, forexample, a TCRα chain and TCRβ chain.

As used herein, the term “arranged in tandem” refers to the arrangementof the genes contiguously, one following or behind the other, in asingle file on a nucleic acid sequence. The genes are ligated togethercontiguously on a nucleic acid sequence, with the coding strands (sensestrands) of each gene ligated together on a nucleic acid sequence.

As used herein, the term “sense strand” refers to the DNA strand of agene that is translated or translatable into protein. When a gene isoriented in the “sense direction” with respect to the promoter in anucleic acid sequence, the “sense strand” is located at the 5′ enddownstream of the promoter, wherein the first codon of the nucleic acidencoding the protein is proximal to the promoter and the last codon isdistal from the promoter.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle, and encodes atleast an exogenous nucleic acid. The vector and/or particle can beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art. The term “virion” is used to refer to a single infective viralparticle. “Viral vector”, “viral vector particle” and “viral particle”also refer to a complete virus particle with its DNA or RNA core andprotein coat as it exists outside the cell. For example, a viral vectormay be selected from adenoviruses, poxviruses, alphaviruses,arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses,herpesviruses, paramyxoviruses, or picornaviruses.

The terms “T cell” or “T lymphocyte” are art-recognized and are intendedto include thymocytes, naïve T lymphocytes, immature T lymphocytes,mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.Illustrative populations of T cells suitable for use in particularembodiments include, but are not limited to, helper T cells (HTL; CD4+ Tcell), a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4−CD8−T cell, natural killer T cell, γδ T cells, or any other subset of Tcells. Other illustrative populations of T cells suitable for use inparticular embodiments include, but are not limited to, T cellsexpressing one or more of the following markers: CD3, CD4, CD8, CD27,CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR and if desired,can be further isolated by positive or negative selection techniques.

The term “statin,” “vastatin,” or as used interchangeably herein“3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor”refers to a pharmaceutical agent which inhibits the enzyme3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzymeis involved in the conversion of HMG-CoA to mevalonate, which is one ofthe steps in cholesterol biosynthesis. Such inhibition is readilydetermined according to standard assays well known to those skilled inthe art.

Preferred statins which may be used in accordance with this presentdisclosure include atorvastatin, disclosed in U.S. Pat. No. 4,681,893;atorvastatin calcium, disclosed in U.S. Pat. No. 5,273,995;cerivastatin, disclosed in U.S. Pat. No. 5,502,199; dalvastatin,disclosed in U.S. Pat. No. 5,316,765; fluindostatin, disclosed in U.S.Pat. No. 4,915,954; fluvastatin, disclosed in U.S. Pat. No. 4,739,073;lovastatin, disclosed in U.S. Pat. No. 4,231,938; mevastatin, disclosedin U.S. Pat. No. 3,983,140; pravastatin, disclosed in U.S. Pat. No.4,346,227; simvastatin, disclosed in U.S. Pat. No. 4,444,784;velostatin, disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171; androsuvastatin, disclosed in U.S. Pat. Nos. 6,858,618 and 7,511,140. Thecontents of each of these patents are hereby incorporated by referencein their entireties. Preferred 3-hydroxy-3-methylglutaryl coenzyme Areductase inhibitors may include atorvastatin, atorvastatin calcium,also known as Liptor®, lovastatin, also known as Mevacor®, pravastatin,also known as Pravachol®, simvastatin, also known as Zocor®, androsuvastatin.

Post-Transcriptional Regulatory Elements

The Woodchuck hepatitis virus (WHV) post-transcriptional regulatoryelement (WPRE) can enhance expression from a number of different vectortypes including lentiviral vectors (U.S. Pat. Nos. 6,136,597; 6,287,814;Zufferey, R., et al. (1999). J. Virol. 73:2886-92). Without wishing tobe bound by theory, this enhancement is thought to be due to improvedRNA processing at the post-transcriptional level, resulting in increasedlevels of nuclear transcripts. A two-fold increase in mRNA stabilityalso contributes to this enhancement (Zufferey, R., et al. ibid). Thelevel of enhancement of protein expression from transcripts containingthe WPRE versus those without the WPRE has been reported to be around2-to-5 fold, and correlates well with the increase in transcript levels.This has been demonstrated with a number of different transgenes(Zufferey, R., et al. ibid).

The WPRE contains three cis-acting sequences important for its functionin enhancing expression levels. In addition, it contains a fragment ofapproximately 180 bp comprising the 5′-end of the WHVX protein ORF (fulllength ORF is 425 bp), together with its associated promoter.Translation from transcripts initiated from the X promoter results information of a protein representing the NH₂-terminal 60 amino acids ofthe X protein. This truncated X protein can promote tumorigenesis,particularly if the truncated X protein sequence is integrated into thehost cell genome at specific loci (Balsano, C. et al., (1991) Biochem.Biophys Res. Commun. 176:985-92; Flajolet, M. et al. (1998) J. Virol.72: 6175-80; Zheng, Y. W., et al. (1994) J. Biol. Chem. 269: 22593-8:Runkel, L., et al. (1993) Virology 197: 529-36). Therefore, expressionof the truncated X protein could promote tumorigenesis if delivered tocells of interest, precluding safe use of wild-type WPRE sequences.

As used herein, the “X region” of the WPRE is defined as comprising atleast the first 60-amino acids of the X protein ORF, including thetranslation initiation codon, and its associated promoter. An “Xprotein” is defined herein as a truncated X protein encoded by an Xprotein ORF as described herein.

The present inventors have introduced mutations into the WPRE sequenceto prevent expression of an X protein. In some aspects, these mutationsare introduced into one or more start codons occurring with the WPREsequence. In some aspects, the X protein promoter and ORF are deletedfrom the WPRE sequence, resulting in a truncated WPRE sequence. Inanother aspect, the X protein promoter and X protein start codon ismutated.

As used herein, a “mutation” can comprise one or more nucleotidedeletions, additions, or substitutions.

In some aspects, the mutated WPRE sequence contains a mutation at one ormore of the start codons corresponding to nucleotide positions 106-108,152-154, 245-247, 272-274, 283-285, 362-364, and 603-605 within the WTWPRE nucleotide sequence according to SEQ ID NO: 1. In some aspects, themutated WPRE sequence contains a mutation at one, at two, at three, atfour, at five, at six, or at all seven of the start codons correspondingto nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285,362-364, and 603-605 within the WT WPRE nucleotide sequence according toSEQ ID NO: 1. In some aspects, the mutated WPRE sequence contains amutation at each of the start codons corresponding to nucleotidepositions 106-108, 152-154, 245-247, 272-274, 283-285, 362-364, and603-605 within the WT WPRE nucleotide sequence according to SEQ ID NO:1.

In another aspect, the mutated WPRE sequence contains a mutation at oneor more of the start codons corresponding to nucleotide positions 70-72,108-110, 121-123, 138-140, 187-189, and 428-430 within the WT WPREnucleotide sequence according to SEQ ID NO: 2. In some aspects, themutated WPRE sequence contains a mutation at one, at two, at three, atfour, at five, or at all six of the start codons corresponding tonucleotide positions 70-72, 108-110, 121-123, 138-140, 187-189, and428-430 within the WT WPRE nucleotide sequence according to SEQ ID NO:2. In some aspects, the mutated WPRE sequence contains a mutation ateach of the start codons corresponding to nucleotide positions 70-72,108-110, 121-123, 138-140, 187-189, and 428-430 within the WT WPREnucleotide sequence according to SEQ ID NO: 2.

The one or more start codon(s) may be mutated at one, two, or all threepositions within the start codon. If more than one start codon ismutated, each start codon mutation may be independent of the others. Inother words, each start codon mutated within the WPRE need not bemutated in an identical manner. In some embodiments, each of the one ormore start codon(s) is mutated at one position within the startcodon(s). For example, the first nucleotide of the start codon may bemutated from “A” to “C”, “G”, or “T”; or the second nucleotide of thestart codon may be mutated from “T” to “A”, “C”, or “G”, or the thirdnucleotide of the start codon may be mutated from “G” to “A”, “C”, or“T”.

In some embodiments, each of the one or more start codon(s) is mutatedat two or at all three positions within the start codon(s). For example,the first nucleotide of the start codon may be mutated from “A” to “C”,“G”, or “T”; and/or the second nucleotide of the start codon may bemutated from “T” to “A”, “C”, or “G”, and/or the third nucleotide of thestart codon may be mutated from “G” to “A”, “C”, or “T”.

In some embodiments, one or more of the start codon(s) is mutated from“ATG” to “TTG”. In some embodiments, each of the one or more startcodon(s) is mutated from “ATG” to “TTG”.

In an aspect, the mutant WPRE sequence is selected from SEQ ID NO: 3 andSEQ ID NO: 4. In another aspect, the mutant WPRE sequence is at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identical to SEQ ID NO: 3 or 4. In some aspects, the mutantWPRE sequence is 95% or more, 96% or more, 97% or more, 98% or more, 98%or more, 99% or more, or 100% identical to SEQ ID NO: 3, wherein themutant WPRE sequence does not comprise “ATG”. In some aspects, themutant WPRE sequence is 95% or more, 96% or more, 97% or more, 98% ormore, 98% or more, 99% or more, or 100% identical to SEQ ID NO: 3,wherein the mutant WPRE sequence does not comprise “ATG” except atnucleotide positions 65-67.

In some aspects, the WPRE sequence is 95% or more, 96% or more, 97% ormore, 98% or more, 98% or more, 99% or more, or 100% identical to SEQ IDNO: 4, wherein nucleotide positions 413-417 are “ATCAT” and nucleotidepositions 428-430 are not “ATG”.

Retroviruses

The concept of using viral vectors for gene or cell therapy isrecognized in, for example, Verma and Somia (1997) Nature 389:239-242,the content of which is incorporated by its entirety.

In an aspect, viruses refer to natural occurring viruses as well asartificial viruses. Viruses in accordance to some embodiments of thepresent disclosure may be either an enveloped or non-enveloped virus.Parvoviruses (such as AAVs) are examples of non-enveloped viruses. In apreferred embodiment, the viruses may be enveloped viruses. In preferredembodiments, the viruses may be retroviruses and in particularlentiviruses. Viral envelope proteins that can promote viral infectionof eukaryotic cells may include HIV-1 derived lentiviral vectors (LVs)pseudotyped with envelope glycoproteins (GPs) from the vesicularstomatitis virus (VSV-G), the modified feline endogenous retrovirus(RD114TR) (SEQ ID NO: 95), and the modified gibbon ape leukemia virus(GALVTR). These envelope proteins can efficiently promote entry of otherviruses, such as parvoviruses, including adeno-associated viruses (AAV),thereby demonstrating their broad efficiency. For example, other viralenvelope proteins may be used including Moloney murine leukemia virus(MLV) 4070 env (such as described in Merten et al., J. Virol.79:834-840, 2005; the content of which is incorporated herein byreference), RD114 env, chimeric envelope protein RD114pro or RDpro(which is an RD114-HIV chimera that was constructed by replacing the Rpeptide cleavage sequence of RD114 with the HIV-1 matrix/capsid (MA/CA)cleavage sequence, such as described in Bell et al. Experimental Biologyand Medicine 2010; 235: 1269-1276; the content of which is incorporatedherein by reference), baculovirus GP64 env (such as described in Wang etal. J. Virol. 81:10869-10878, 2007; the content of which is incorporatedherein by reference), or GALV env (such as described in Merten et al.,J. Virol. 79:834-840, 2005; the content of which is incorporated hereinby reference), or derivatives thereof.

As used herein, the term “retrovirus” includes, but is not limited to,murine leukemia virus (MLV), human immunodeficiency virus (HIV), equineinfectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV) and all other retroviridiae includinglentiviruses.

A detailed list of retroviruses may be found in Coffin et al.(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviruses also belong to the retrovirus family, but they can infectboth dividing and non-dividing cells.

The lentivirus group can be split into “primate” and “non-primate”.Examples of primate lentiviruses include the human immunodeficiencyvirus (HIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis encephalitis virus (CAEV), equine infectious anemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

Details on the genomic structure of some lentiviruses may be found, forexample, in the NCBI Genbank database (i.e. GenBank Accession Nos.AF033819 and AF033820 respectively). Details of HIV variants may also befound in the HIV databases maintained by Los Alamos National Laboratory.

During the process of infection, a retrovirus initially attaches to aspecific cell surface receptor. On entry into the susceptible host cell,the retroviral RNA genome is then copied to DNA by the virally encodedreverse transcriptase, which is carried inside the parent virus. ThisDNA is transported to the host cell nucleus where it subsequentlyintegrates into the host genome. At this stage, it is typically referredto as the provirus. The provirus is stable in the host chromosome duringcell division and is transcribed like other cellular genes. The provirusencodes the proteins and other factors required to make more virus,which can leave the cell by a process sometimes called “budding”.

Each retroviral genome comprises genes called gag, pol and env, whichcode for virion proteins and enzymes. These genes are flanked at bothends by regions called long terminal repeats (LTRs). The LTRs areresponsible for proviral integration, and transcription. They also serveas enhancer-promoter sequences. In other words, the LTRs can control theexpression of the viral genes. Encapsidation of the retroviral RNAsoccurs by virtue of a psi sequence located at the 5′ end of the viralgenome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R, and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′ end of the RNA. The sizes of the three elements canvary considerably among different retroviruses.

For the viral genome, the site of transcription initiation is at theboundary between U3 and R in the left hand side LTR and the site of poly(A) addition (termination) is at the boundary between R and U5 in theright hand side LTR. U3 contains most of the transcriptional controlelements of the provirus, which include the promoter and multipleenhancer sequences responsive to cellular and in some cases, viraltranscriptional activator proteins. Some retroviruses have any one ormore of the following genes that code for proteins that are involved inthe regulation of gene expression: tat, rev, tax and rex.

With regard to the structural genes gag, pol and env themselves; gagencodes the internal structural protein of the virus. Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome. The envgene encodes the surface (SU) glycoprotein and the transmembrane (TM)protein of the virion, which form a complex that interacts specificallywith cellular receptor proteins. This interaction leads ultimately toinfection by fusion of the viral membrane with the cell membrane.

Retroviruses may also contain “additional” genes, which code forproteins other than gag, pol and env. Examples of additional genesinclude, in HIV, one or more of vif, vpr, vpx, vpu, tat, rev, and nef.EIAV contains, for example, the additional genes S2 and dUTPase.

Proteins encoded by additional genes serve various functions, some ofwhich may be duplicative of a function provided by a cellular protein.In EIAV, for example, tat acts as a transcriptional activator of theviral LTR. It binds to a stable, stem-loop RNA secondary structurereferred to as TAR. Rev regulates and co-ordinates the expression ofviral genes through rev-response elements (RRE). The mechanisms ofaction of these two proteins are thought to be broadly similar to theanalogous mechanisms in the primate viruses. The function of S2 isunknown but it does not appear to be essential. In addition, an EIAVprotein, Ttm, has been identified that is encoded by the first exon oftat spliced to the env coding sequence at the start of the transmembraneprotein.

Delivery Systems

Retroviral vector systems have been proposed as a delivery system for,inter alia, the transfer of a nucleotide of interest (NOI) to one ormore sites of interest. The transfer can occur in vitro, ex vivo, invivo, or combinations thereof. Retroviral vector systems have even beenexploited to study various aspects of the retrovirus life cycle,including receptor usage, reverse transcription and RNA packaging(reviewed by Miller, 1992 Curr Top Microbiol Immunol 158:1-24, thecontent which is incorporated herein by reference).

A recombinant retroviral vector particle is capable of transducing arecipient cell with an NOI. Once within the cell, the RNA genome fromthe vector particle is reverse transcribed into DNA and integrated intothe DNA of the recipient cell.

As used herein, the term “vector genome” refers to the RNA constructpresent in the retroviral vector particle and/or the integrated DNAconstruct. The term also embraces a separate or isolated DNA constructcapable of encoding such an RNA genome. A retroviral or lentiviralgenome should comprise at least one component part derivable from aretrovirus or a lentivirus. The term “derivable” is used in its normalsense as meaning a nucleotide sequence or a part thereof, which need notnecessarily be obtained from a virus such as a lentivirus but insteadcould be derived therefrom. By way of example, the sequence may beprepared synthetically or by use of recombinant DNA techniques.Preferably, the genome comprises a psi region (or an analogous componentthat is capable of causing encapsidation).

The viral vector genome is preferably “replication defective”, by whichwe mean that the genome does not comprise sufficient genetic informationalone to enable independent replication to produce infectious viralparticles within the recipient cell. In a preferred embodiment, thegenome lacks a functional env, gag or pol gene.

The viral vector genome may comprise some or all of the long terminalrepeats (LTRs). Preferably, the genome comprises at least part of theLTRs or an analogous sequence, which is capable of mediating proviralintegration, and transcription. The sequence may also comprise or act asan enhancer-promoter sequence.

The viral vector genome according to some aspects of the invention maybe provided as a kit of parts. For example, the kit may comprise (i) aplasmid or plasm ids containing the NOIs and internal regulatorysequences, such as, for example, a promoter or an IRES sequence(s); and(ii) a retroviral genome construct with suitable restriction enzymerecognition sites for cloning the NOIs and internal regulatorysequence(s) into the viral genome.

It is recognized that the separate expression of the components requiredto produce a retroviral vector particle on separate DNA sequencesco-introduced into the same cell will yield retroviral particlescarrying defective retroviral genomes that carry therapeutic genes. Thiscell is referred to as the producer cell.

There are two common procedures for generating producer cells. In one,the sequences encoding retroviral Gag, Pol and Env proteins areintroduced into the cell and stably integrated into the cell genome; astable cell line is produced which is referred to as the packaging cellline. The packaging cell line produces the proteins required forpackaging retroviral RNA but it cannot bring about encapsidation due tothe lack of a psi region. However, when a vector genome having a psiregion is introduced into the packaging cell line, the helper proteinscan package the psi-positive recombinant vector RNA to produce therecombinant virus stock. This can be used to transduce the NOI intorecipient cells. The recombinant virus whose genome lacks all genesrequired to make viral proteins can infect only once and cannotpropagate. Hence, the NOI is introduced into the host cell genomewithout the generation of potentially harmful retrovirus. A summary ofthe available packaging lines is presented in “Retroviruses” (1997 ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 449, the content which is incorporated by reference in its entirety).

The present invention also provides a packaging cell line comprising aviral vector genome of the present invention. For example, the packagingcell line may be transduced with a viral vector system comprising thegenome or transfected with a plasmid carrying a DNA construct capable ofencoding the RNA genome. The present invention also provides aretroviral (or lentiviral) vector particle produced by such a cell.

The second approach is to introduce the three different DNA sequencesthat are required to produce a retroviral vector particle, i.e. the envcoding sequences, the gag-pol coding sequence and the defectiveretroviral genome containing one or more NOIs into the cell at the sametime by transient transfection and the procedure is referred to astransient triple transfection (Landau & Littman 1992; Pear et al. 1993).The triple transfection procedure has been optimized (Soneoka et al.1995; Finer et al. 1994). WO 94/29438 describes the production ofproducer cells in vitro using this multiple DNA transient transfectionmethod.

The components of the viral system, which are required to complement thevector genome, may be present on one or more “producer plasmids” fortransfecting into cells.

The present invention also provides a vector system for producing aretrovirus-derived particle, comprising (i) a retroviral genomeaccording to some aspects of the invention; (ii) a nucleotide sequencecoding for retroviral gag and pol proteins; (iii) nucleotide sequencesencoding other essential viral packaging components not encoded by thenucleotide sequence of (ii).

In an aspect, the nucleic acid sequence(s) encoding at least one of Vpr,Vif, Tat, Nef, or analogous auxiliary genes, from the retrovirus fromwhich the particles are derived, are disrupted such as said nucleic acidsequence(s) are incapable of encoding functional Vpr, Vif, Tat, Nef, oranalogous auxiliary proteins, or are removed from the system.

The present invention also provides a cell transfected with such avector system and a retroviral vector particle produced by such a cell.Preferably, the gag-pol sequence is codon optimized for use in theparticular producer cell (see below).

The env protein encoded by the nucleotide sequence of iii) may be ahomologous retroviral or lentiviral env protein. Alternatively, it maybe a heterologous env, or an env from a non-retro or lentivirus (seebelow under “pseudotyping”).

The term “viral vector system” is used generally to mean a kit of partsthat can be used when combined with other necessary components for viralparticle production to produce viral particles in host cells. Forexample, the retroviral vector genome may lack one or more of the genesneeded for viral replication. This may be combined in a kit with afurther complementary nucleotide sequence or sequences, for example onone or more producer plasm ids. By co-transfection of the genometogether with the producer plasm id(s), the necessary components shouldbe provided for the production of infectious viral particles.

Alternatively, the complementary nucleotide sequence(s) may be stablypresent within a packaging cell line that is included in the kit.

The present invention also relates to a retroviral vector system, whichis capable of delivering an RNA genome to a recipient cell, wherein thegenome is longer than the wild type genome of the lentivirus.

In some aspects, the RNA genome of the vector system has up to 5%,preferably, up to 10% or even up to 30% more bases than the wild-typegenome. Preferably, the RNA genome is about 10% longer than thewild-type genome. For example, wild type EIAV comprises an RNA genome ofapproximately 8 kb. An EIAV vector system of the present invention mayhave an RNA genome of up to (preferably about) 8.8 kb.

In some aspects, the retroviral vector system of the present inventionis a self-inactivating (SIN) vector system. For example,self-inactivating retroviral vector systems have been constructed bydeleting the transcriptional enhancers or the enhancers and promoter inthe U3 region of the 3′ LTR. After a round of vector reversetranscription and integration, these changes are copied into both the 5′and the 3′ LTRs, producing a transcriptionally inactive provirus.However, any promoter(s) internal to the LTRs in such vectors will stillbe transcriptionally active. This strategy has been employed toeliminate effects of the enhancers and promoters in the viral LTRs ontranscription from internally placed genes. Such effects includeincreased transcription or suppression of transcription. This strategycan also be used to eliminate downstream transcription from the 3′ LTRinto genomic DNA. This is of particular concern in human gene therapywhere it may be important to prevent the adventitious activation of anendogenous oncogene.

In some aspects, a recombinase-assisted mechanism is used, whichfacilitates the production of high titer regulated lentiviral vectorsfrom the producer cells of the present invention.

In some aspects, the present disclosure comprises a method oftransducing T cells comprising: obtaining T cells from at least onedonor, patient, or individual; activating the T cells with an anti-CD3antibody and/or an anti-CD28 antibody; transducing the activated T cellswith a viral vector; and optionally expanding the transduced T cells;optionally measuring a quantity of the expanded T cells that express thetransgene and/or a copy number of integrated transgene in each of the Tcells at the plurality of volumetric concentrations; and optionallyidentifying the volumetric concentration that yields a maximum averageof the quantity of the expanded T cells that express the transgeneand/or a maximum average of the copy number of the integrated transgenewithout exceeding five copies of the integrated transgene in each of theexpanded T cells from the plurality of healthy donors, and transducing Tcells obtained from a patient with the viral vector at the identifiedvolumetric concentration for the immunotherapy, as described inUS20190216852 (the content of which is hereby incorporated by referencein its entirety).

In some aspects, the plurality of volumetric concentrations are fromabout 0.01 μl per about 10⁶ cells to about 1 ml per about 10⁶ cells;from about 0.01 μl per about 2×10⁶ cells to about 1 ml per about 2×10⁶cells; from about 0.01 μl per about 5×10⁶ cells to about 1 ml per about5×10⁶ cells; from about 0.01 μl per about 10⁷ cells to about 1 ml perabout 10⁷ cells; from about 1 μl per about 10⁷ cells to about 500 μl perabout 10⁷ cells; from about 5 μl per about 10⁷ cells to about 150 μl perabout 10⁷ cells; or from about 8 μl per about 10⁷ cells to about 12 μlper about 10⁷ cells.

As used herein, the term “recombinase assisted system” includes, but isnot limited to, a system using the Cre recombinase/loxP recognitionsites of bacteriophage P1 or the site-specific FLP recombinase of S.cerevisiae, which catalyzes recombination events between 34 bp FLPrecognition targets (FRTs).

The site-specific FLP recombinase of S. cerevisiae, which catalyzesrecombination events between 34 bp FLP recognition targets (FRTs), hasbeen configured into DNA constructs to generate high level producer celllines using recombinase-assisted recombination events (Karreman et al.(1996) NAR 24:1616-1624). A similar system has been developed using theCre recombinase/loxP recognition sites of bacteriophage P1 (Vanin et al.(1997) J. Virol 71:7820-7826). This was configured into a lentiviralgenome such that high titer lentiviral producer cell lines weregenerated.

By using producer/packaging cell lines, it is possible to propagate andisolate quantities of retroviral vector particles (e.g. to preparesuitable titers of the retroviral vector particles) for subsequenttransduction of, for example, a site of interest (such as a specificorgan or tissue) or in a cell of interest (such as a T cell). Producercell lines are usually better for large-scale production of vectorparticles.

Transient transfection has certain advantages over the packaging cellmethod. In this regard, transient transfection avoids the longer timerequired to generate stable vector-producing cell lines and is used ifthe vector genome or retroviral packaging components are toxic to cells.If the vector genome encodes toxic genes or genes that interfere withthe replication of the host cell, such as inhibitors of the cell cycleor genes that induce apoptosis, it may be difficult to generate stablevector-producing cell lines, but transient transfection can be used toproduce the vector before the cells die. Also, cell lines have beendeveloped using transient transfection that produce vector titer levelsthat are comparable to the levels obtained from stable vector-producingcell lines (Pear et al. 1993, PNAS 90:8392-8396).

Producer cells/packaging cells can be of any suitable cell type.Producer cells are generally mammalian cells, but can be, for example,insect cells.

As used herein, the term “producer cell” or “vector producing cell”refers to a cell that contains all the elements necessary for productionof retroviral vector particles.

In some aspects, the producer cell is obtainable from a stable producercell line, from a derived stable producer cell line, or from a derivedproducer cell line.

As used herein, the term “derived producer cell line” is a transducedproducer cell line that has been screened and selected for highexpression of a marker gene. Such cell lines support high-levelexpression from the retroviral genome. The term “derived producer cellline” is used interchangeably with the term “derived stable producercell line” and the term “stable producer cell line”.

In some aspects, the derived producer cell line includes, but is notlimited to, a retroviral and/or a lentiviral producer cell.

In some aspects, the envelope protein sequences, and nucleocapsidsequences are all stably integrated in the producer and/or packagingcell. However, one or more of these sequences could also exist inepisomal form and gene expression could occur from the episome.

As used herein, the term “packaging cell” refers to a cell that containsthose elements necessary for production of infectious recombinant virusthat are lacking in the RNA genome. Typically, such packaging cellscontain one or more producer plasmids, which are capable of expressingviral structural proteins (such as codon optimized gag-pol and env) butthey do not contain a packaging signal.

The term “packaging signal” which is referred to interchangeably as“packaging sequence” or “psi” is used in reference to the non-coding,cis-acting sequence required for encapsidation of retroviral RNA strandsduring viral particle formation. In HIV-1, this sequence has been mappedto loci extending from upstream of the major splice donor site (SD) toat least the gag start codon.

Packaging cell lines suitable for use with the above-described vectorconstructs may be readily prepared (see also WO 92/05266, the content ofwhich is incorporated by reference), and utilized to create producercell lines for the production of retroviral vector particles. Asmentioned above, a summary of the available packaging lines is presentedin “Retroviruses”.

Also, as discussed above, simple packaging cell lines, comprising aprovirus in which the packaging signal has been deleted, have been foundto lead to the rapid production of undesirable replication competentviruses through recombination. In order to improve safety,second-generation cell lines have been produced, wherein the 3′ LTR ofthe provirus is deleted. In such cells, two recombinations would benecessary to produce a wild type virus. A further improvement involvesthe introduction of the gag-pol genes and the env gene on separateconstructs, so-called third generation packaging cell lines. Theseconstructs are introduced sequentially to prevent recombination duringtransfection.

In some aspects, the packaging cell lines are second-generationpackaging cell lines or third generation packaging cell lines.

In these split-construct, third generation cell lines, a furtherreduction in recombination may be achieved by changing the codons. Thistechnique, based on the redundancy of the genetic code, aims to reducehomology between the separate constructs, for example, between theregions of overlap in the gag-pol and env open reading frames.

The packaging cell lines are useful for providing the gene productsnecessary to encapsulate and provide a membrane protein for a high titervector particle production. The packaging cell may be a cell cultured invitro, such as a tissue culture cell line. Suitable cell lines include,but are not limited to, mammalian cells, such as murine fibroblastderived cell lines or human cell lines. In some aspects, the packagingcell line is a primate or human cell line, such as for example: HEK293,293-T, TE671, HT1080.

It is desirable to use high-titer virus preparations in bothexperimental and practical applications. Techniques for increasing viraltiter include using a psi plus packaging signal as discussed above andconcentration of viral stocks.

As used herein, the term “high titer” means an effective amount of aretroviral vector or particle that is capable of transducing a targetsite such as a cell.

As used herein, the term “effective amount” means an amount of aretroviral or lentiviral vector or vector particle that is sufficient toinduce expression of the NOIs at a target site.

A high-titer viral preparation for a producer/packaging cell is usuallyon the order of 10⁵ to 10⁷ retrovirus particles per mL. In anotheraspect, the preparation has at least 10⁸ TU/mL, preferably from 10⁸ to10⁹ TU/mL, more preferably at least 10⁹ TU/mL (titer is expressed intransducing units per mL (TU/mL) as titred on a standard D17 cell line).Other methods of concentration such as ultrafiltration or binding to andelution from a matrix may be used.

The expression products encoded by the NOIs may be proteins that aresecreted from the cell. Alternatively, the NOI expression products arenot secreted and are active within the cell. For some applications, itis preferred for the NOI expression product to demonstrate a bystandereffect or a distant bystander effect; that is the production of theexpression product in one cell leading to the modulation of additional,related cells, either neighboring or distant (e.g. metastatic), whichpossess a common phenotype (Zennou et al., (2000) Cell 101: 173;Folleuzi et al., (2000) Nat. Genetics 25: 217; Zennou et al., (2001)Nat. Biotechnol. 19: 446), the content of each which is incorporated byreference in their entireties.

The presence of a sequence termed the central polypurine tract (cPPT)may improve the efficiency of gene delivery to non-dividing cells. Thiscis-acting element is located, for example, in the viral polymerasecoding region element. In some aspects, the viral genome of the presentinvention comprises a cPPT sequence.

In addition, the viral genome may comprise a translational enhancer.

The NOIs may be operatively linked to one or more promoter/enhancerelements. Transcription of one or more NOIs may be under the control ofviral LTRs or alternatively promoter-enhancer elements. In some aspects,the promoter is a strong viral promoter such as CMV, or is a cellularconstitutive promoter such as PGK, beta-actin or EFlalpha. The promotermay be regulated or tissue-specific. The control of expression can alsobe achieved by using such systems as the tetracycline system thatswitches gene expression on or off in response to outside agents (forexample, tetracycline or its analogues).

Pseudotyping

In the design of retroviral vector systems, it is desirable to engineerparticles with different target cell specificities to the native virus,to enable the delivery of genetic material to an expanded or alteredrange of cell types. One manner in which to achieve this is byengineering the virus envelope protein to alter its specificity. Anotherapproach is to introduce a heterologous envelope protein into the vectorparticle to replace or add to the native envelope protein of the virus.

The term pseudotyping means incorporating in at least a part of, orsubstituting a part of, or replacing all of an env gene of a viralgenome with a heterologous env gene, for example, an env gene fromanother virus. Pseudotyping is not a new phenomenon and examples may befound in WO 99/61639, WO-A-98/05759, WO-A-98/05754, WO-A-97/17457,WO-A-96/09400, WO-A-91/00047 and Mebatsion et al. (1997) Cell 90:841-847, the content of each which is herein in incorporated byreference in their entireties.

In some aspects, the vector system is pseudotyped with a gene encodingat least part of the rabies G protein. Examples of rabies G pseudotypedretroviral vectors may be found in WO99/61639. In a further aspect, thevector system is pseudotyped with a gene encoding at least part of theVSV-G protein. Examples of VSV-G pseudotyped retroviral vectors may befound in U.S. Pat. No. 5,817,491, the content of which is hereinincorporated by reference in its entirety. In another aspect, the vectoris pseudotyped with an envelope protein of a virus selected from thenative feline endogenous virus (RD114), a chimeric version of RD114(RD114TR; SEQ ID NO: 95), gibbon ape leukemia virus (GALV), a chimericversion of GALV (GALV-TR), amphotropic murine leukemia virus (MLV4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowlplague virus (FPV), Ebola virus (EboV), baboon retroviral envelopeglycoprotein (BaEV), or lymphocytic choriomeningitis virus (LCMV).

It has been demonstrated that a retrovirus or lentivirus minimal systemcan be constructed from HIV, SIV, FIV, and EIAV viruses. Such a systemrequires none of the additional genes vif, vpr, vpx, Vpu, tat, rev andnef for either vector production or for transduction of dividing andnon-dividing cells. It has also been demonstrated that an EIAV minimalvector system can be constructed which does not require S2 for eithervector production or for transduction of dividing and non-dividingcells. The deletion of additional genes is advantageous. Firstly, itpermits vectors to be produced without the genes associated with diseasein lentiviral (e.g. HIV) infections. In particular, tat is associatedwith disease. Secondly, the deletion of additional genes permits thevector to package more heterologous DNA. Thirdly, genes whose functionis unknown, such as S2, may be omitted, thus reducing the risk ofcausing undesired effects. Examples of minimal lentiviral vectors aredisclosed in WO-A-99732646 and in WO-A-98/17815, the content of which isherein incorporated by reference in its entirety.

The absence of functional auxiliary genes from the retroviral vectorproduction system means that those functional genes will also be absentfrom retroviral vector particles produced by the system. Also, anyauxiliary proteins that would otherwise be encoded by those genes andincorporated into the vector particles will be absent from the vectorparticles. In known retroviral vector production systems, the auxiliarygenes may be present as part of the vector genome encoding DNA, ortogether with the packaging components. The location of an auxiliarygene in a vector production system depends in part on its relationshipwith other retroviral components. For example, vif is often part of agag-pol packaging cassette in a packaging cell. Thus, to remove afunctional auxiliary gene for the purposes of the invention may involveits removal from the packaging components, or from the vector genome, orperhaps both.

To remove a functional auxiliary gene may not require removal of thegene in its entirety. Usually removal of part of the gene, or disruptionof the gene in some other way will be sufficient. The absence of afunctional auxiliary gene is understood herein to mean that the gene isnot present in a form in which it is capable of encoding the functionalauxiliary protein.

In some aspects, functional vpr and tat genes or analogous genesnormally present in the lentivirus on which the vector particles arebased are both absent. These two auxiliary genes are associated withcharacteristics of lentiviruses that are particularly undesirable for agene or cell therapy vector. However, other than by the proviso givenabove, the invention is not limited with regard to the combination ofauxiliary genes that are absent in a system according to the inventionfor producing HIV-1-based vector particles, any combination of three, ormore preferably four, of the genes may be absent in their functionalform. Most preferably, all five of the auxiliary genes vpr, vif, tat,nef, and vpu are absent in their functional form. Similarly, for systemsconcerned with other lentiviruses, it is most preferable that all of theauxiliary genes are absent in their functional form (except rev which ispreferably present unless replaced by a system analogous to the rev/RREsystem).

Thus, in some aspects, the delivery system according to the invention isdevoid of at least tat and S2 (if it is an EIAV vector system), andpossibly also vif, vpr, vpx, vpu and nef. Preferably, the systems of thepresent invention are also devoid of rev. Rev was previously thought tobe essential in some retroviral genomes for efficient virus production.For example, in the case of HIV, it was thought that rev and RREsequence should be included. However, it has been found that therequirement for rev and RRE can be reduced or eliminated by codonoptimization (see below) or by replacement with other functionalequivalent systems such as the MPMV system. As expression of thecodon-optimized gag-pol is rev-independent, RRE can be removed from thegag-pol expression cassette, thus removing any potential forrecombination with any RRE contained on the vector genome.

In some aspects, the viral genome of the present invention lacks the Revresponse element (RRE). In another aspect, a nucleic acid sequenceencoding Rev, or a functional equivalent thereof, is disrupted such thatthe nucleic acid sequence is incapable of encoding the functional Rev oris removed from the vector genome.

In some aspects, the system used in the present invention is based on aso-called “minimal system in which some or all of the additional geneshave been removed. Preferably, the viral vector of the present inventionhas a minimal viral genome.

As used herein, the term “minimal viral genome” means that the viralvector has been manipulated so as to remove the non-essential elementsand to retain the essential elements to provide the requiredfunctionality to infect, transduce and deliver a NOI to a target hostcell. Preferably, the viral vector with the minimal viral genome is aminimal lentiviral vector.

Codon Optimization

Codon optimization has previously been described in WO 99/41397, thecontent of which is herein incorporated by reference in its entirety.Different cells differ in their usage of particular codons. This codonbias corresponds to a bias in the relative abundance of particular tRNAsin the cell type. By altering the codons in the sequence to match withthe relative abundance of corresponding tRNAS, it is possible toincrease expression. By the same token, it is possible to decreaseexpression by deliberately choosing codons for which the correspondingtRNAs are known to be rare in the particular cell type. Thus, anadditional degree of translational control is available.

Many viruses, including HIV and other lentiviruses, use a large numberof rare codons and by changing these to correspond to commonly usedmammalian codons, increased expression of the packaging components inmammalian producer cells can be achieved. Codon usage tables are knownin the art for mammalian cells, as well as for a variety of otherorganisms.

Codon optimization has a number of other advantages. By virtue ofalterations in their sequences, the nucleotide sequences encoding thepackaging components of the viral particles required for assembly ofviral particles in the producer cells/packaging cells have RNAinstability sequences (INS) eliminated from them. At the same time, theamino acid sequence coding sequence for the packaging components isretained so that the viral components encoded by the sequences remainthe same, or at least sufficiently similar that the function of thepackaging components is not compromised. Codon optimization alsoovercomes the Rev/RRE requirement for export, rendering optimizedsequences Rev-independent. Codon optimization also reduces homologousrecombination between different constructs within the vector system (forexample, between the regions of overlap in the gag-pol and env openreading frames). The overall effect of codon optimization is therefore anotable increase in viral titer and improved safety.

In one aspect, only codons relating to INS are codon optimized. However,in a more preferred and practical embodiment, the sequences are codonoptimized in their entirety, with the exception of the sequenceencompassing the frameshift site.

The gag-pol gene comprises two overlapping reading frames encoding gagand pol proteins respectively. The expression of both proteins dependson a frameshift during translation. This frameshift occurs as a resultof ribosome “slippage” during translation. This slippage is thought tobe caused at least in part by ribosome-stalling RNA secondarystructures. Such secondary structures exist downstream of the frameshiftsite in the gag-pol gene. For HIV, the region of overlap extends fromnucleotide 1222 downstream of the beginning of gag (wherein nucleotide 1is the A of the gag ATG) to the end of gag (nt 1503). Consequently, a281 bp fragment spanning the frameshift site and the overlapping regionof the two reading frames is preferably not codon optimized. Retainingthis fragment will enable more efficient expression of the gag-polproteins.

Derivations from optimal codon usage may be made, for example, toaccommodate convenient restriction sites, and conservative amino acidchanges may be introduced into the gag-pol proteins.

In some aspects, codon optimization is based on highly expressedmammalian genes. The third and sometimes the second and third base maybe changed.

Due to the degenerate nature of the genetic code, it will be appreciatedthat a skilled worker can achieve numerous gag-pol sequences. Also,there are many retroviral variants described that can be used as astarting point for generating a codon optimized gag-pol sequence.Lentiviral genomes can be quite variable. For example, there are manyquasi-species of HIV-1 that are still functional. This is also the casefor EIAV. These variants may be used to enhance particular parts of thetransduction process. Details of HIV variants may also be found in theHIV databases maintained by Los Alamos National Laboratory. Details ofEIAV clones may be found at the NCBI database maintained by the NationalInstitutes of Health.

The strategy for codon optimized gag-pol sequences can be used inrelation to any retrovirus. This would apply to all lentiviruses,including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. In addition,this method could be used to increase expression of genes from HTLV-1,HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV, andother retroviruses.

Codon optimization can render gag-pol expression Rev independent. Toenable the use of anti-rev or RRE factors in the retroviral vector,however, it would be necessary to render the viral vector generationsystem totally Rev/RRE independent. Thus, the genome also should bemodified. This can be achieved by optimizing vector genome components.Advantageously, these modifications can also lead to the production of asafer system absent of all additional proteins both in the producer andin the transduced cell.

As described above, the packaging components for a retroviral vectorinclude expression products of gag, pol, and env genes. In addition,efficient packaging depends on a short sequence of 4 stem loops followedby a partial sequence from gag and env (the “packaging signal”). Thus,inclusion of a deleted gag sequence in the retroviral vector genome (inaddition to the full gag sequence on the packaging construct) willoptimize vector titer. To date, efficient packaging has been reported torequire from 255 to 360 nucleotides of gag in vectors that still retainenv sequences, or about 40 nucleotides of gag in a particularcombination of splice donor mutation, gag and env deletions. It has beenfound that a deletion of all but the N-terminal 360 nucleotides or so ingag leads to an increase in vector titer. Thus, preferably, theretroviral vector genome includes a gag sequence that comprises one ormore deletions, more preferably the gag sequence comprises about 360nucleotides derivable from the N-terminus.

NOIs

In the present invention, the term NOI (nucleotide sequence of interest)includes any suitable nucleotide sequence, which need not necessarily bea complete naturally occurring DNA or RNA sequence. Thus, the NOI canbe, for example, a synthetic RNA/DNA sequence, a codon optimized RNA/DNAsequence, a recombinant RNA/DNA sequence (i.e. prepared by use ofrecombinant DNA techniques), a cDNA sequence or a partial genomic DNAsequence, including combinations thereof. The sequence need not be acoding region. If it is a coding region, it need not be an entire codingregion. In addition, the RNA/DNA sequence can be in a sense orientationor in an anti-sense orientation. Preferably, it is in a senseorientation. Preferably, the sequence is, comprises, or is transcribedfrom cDNA.

The NOI(s), also referred to as heterologous sequence(s), heterologousgene(s) or transgene(s), may be any heterologous sequence of interestwithout limitation, including, for example, sequences coding fortherapeutic proteins, enzymes, and antibodies, etc.; siRNA; anti-sense;microRNAs, aptamers; ribozymes, any gene inhibitory or silencingsequence; and any sequence which is to be delivered to a host cell via alentiviral transducing vector, such as any one or more of a selectiongene(s), marker gene(s) and therapeutic gene(s).

The NOI may be a candidate gene that is of potential significance in adisease process. Thus, the vector system of the present invention may,for example, be used for target validation purposes.

The NOI may have a therapeutic or diagnostic application. Suitable NOIsinclude, but are not limited to: sequences encoding enzymes, cytokines,chemokines, hormones, antibodies, anti-oxidant molecules, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,immune co-stimulatory molecules, immunomodulatory molecules, anti-senseRNA, small interfering RNA (siRNA), a trans dominant negative mutant ofa target protein, a toxin, a conditional toxin, an antigen, an antigenreceptor, a chimeric antigen receptor, a T-cell receptor, a tumorsuppressor protein, and growth factors, membrane proteins, pro- andanti-angiogenic proteins and peptides, vasoactive proteins and peptides,antiviral proteins and ribozymes, and derivatives thereof (such as withan associated reporter group). The NOIs may also encode pro-drugactivating enzymes. When used in a research context, the NOIs may alsoencode reporter genes such as, but not limited to, green fluorescentprotein (GFP), luciferase, β-galactosidase, or resistance genes toantibiotics such as, for example, ampicillin, neomycin, bleomycin,Zeocin, chloramphenicol, hygromycin, kanamycin, among others.

The NOI may encode all or part of the protein of interest (“POI”), or amutant, homologue or variant thereof. For example, the NOI may encode afragment of the POI that is capable of functioning in vivo in ananalogous manner to the wild-type protein.

The term “mutant” includes POIs that include one or more amino acidvariations from the wild-type sequence. For example, a mutant maycomprise one or more amino acid additions, deletions or substitutions.

Here, the term “homologue” means an entity having a certain homologywith the NOI, or which encodes a protein having a degree of homologywith the POI. Here, the term “homology” can be equated with “identity”.

In an aspect, vectors, constructs, or sequences described herein maycomprise at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% to a reference sequence. A sequence “at least85% identical to a reference sequence” is a sequence having, on itsentire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity with the entire length of thereference sequence. In an aspect, vectors, constructs, or sequencesdescribed herein may comprise at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% to any of SEQ ID NO:1-95.

In the context of the present application, the “percentage of identity”or “percent identity” is calculated using a global pairwise alignment(i.e. the two sequences are compared over their entire length). Methodsfor comparing the identity of two or more sequences are well known inthe art. The «needle» program, which uses the Needleman-Wunsch globalalignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol.48:443-453) to find the optimum alignment (including gaps) of twosequences when considering their entire length, may for example be used.The needle program is for example available on the ebi.ac.uk World WideWeb site and is further described in the following publication (EMBOSS:The European Molecular Biology Open Software Suite (2000) Rice, P.Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). Thepercentage of identity between two polypeptides, in accordance with theinvention, is calculated using the EMBOSS: needle (global) program witha “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to0.5, and a Blosum62 matrix.

Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical” to a reference sequence maycomprise mutations such as deletions, insertions and/or substitutionscompared to the reference sequence. In case of substitutions, theprotein consisting of an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% A identical to a reference sequence maycorrespond to a homologous sequence derived from another species thanthe reference sequence.

“Amino acid substitutions” may be conservative or non-conservative.Preferably, substitutions are conservative substitutions, in which oneamino acid is substituted for another amino acid with similar structuraland/or chemical properties.

In an embodiment, conservative substitutions may include those, whichare described by Dayhoff in “The Atlas of Protein Sequence andStructure. Vol. 5”, Natl. Biomedical Research, the contents of which areincorporated by reference in their entirety. For example, in an aspect,amino acids, which belong to one of the following groups, can beexchanged for one another, thus, constituting a conservative exchange:Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine(S), threonine (T); Group 2: cysteine (C), serine (S), tyrosine (Y),threonine (T); Group 3: valine (V), isoleucine (I), leucine (L),methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K),arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y),tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamicacid (E). In an aspect, a conservative amino acid substitution may beselected from the following of T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G,and/or T→S.

In a further embodiment, a conservative amino acid substitution mayinclude the substitution of an amino acid by another amino acid of thesame class, for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met,Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3)acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative aminoacid substitutions may also be made as follows: (1) aromatic: Phe, Tyr,His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) protonacceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Pat.No. 10,106,805, the contents of which are incorporated by reference intheir entirety).

In another embodiment, conservative substitutions may be made inaccordance with Table A. Methods for predicting tolerance to proteinmodification may be found in, for example, Guo et al., Proc. Natl. Acad.Sci., USA, 101(25):9205-9210 (2004), the contents of which areincorporated by reference in their entirety.

TABLE A Conservative Amino Acid substitution Conservative Amino AcidSubstitutions Amino Acid Substitutions (others are known in the art) AlaSer, Gly, Cys Arg Lys, Gln, His Asn Gln, His, Glu, Asp Asp Glu, Asn, GlnCys Ser, Met, Thr Gln Asn, Lys, Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro,Ala, Ser His Asn, Gln, Lys Ile Leu, Val, Met, Ala Leu Ile, Val, Met, AlaLys Arg, Gln, His Met Leu, Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp,His Ser Thr, Cys, Ala Thr Ser, Val, Ala Trp Tyr, Phe Tyr Trp, Phe, HisVal Ile, Leu, Met, Ala, Thr

In an aspect, sequences described herein may include 1, 2, 3, 4, 5, 10,15, 20, 25, or 30 amino acid or nucleotide mutations, substitutions,deletions. In an aspect, any one of SEQ ID NO: 1-95 may include 1, 2, 3,4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. Inyet another aspect, the mutations or substitutions are conservativeamino acid substitutions.

In another embodiment, conservative substitutions may be those shown inTable B under the heading of “conservative substitutions.” If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table B,may be introduced and the products screened if needed.

TABLE B Amino Acid substitution Amino Acid Substitutions OriginalResidue (naturally occurring amino Conservative acid) SubstitutionsExemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys; Gln;Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) SerSer; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His(H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe;Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) ArgArg; Gln; Asn Met (M) Len Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala;Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; PheTyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;Norleucine

Internal Ribosome Entry Site (IRES)

The viral genome of the present invention comprises at least one, butcan optionally comprise two or more NOIs. In order for two or more NOIsto be expressed, there may be two or more transcription units within thevector genome, one for each NOI. However, it is clear from theliterature that retroviral vectors achieve the highest titers and mostpotent gene expression properties if they are kept genetically simple(PCT/GB96/01230; Bowtell et al., 1988 J. Virol. 62, 2464; Correll etal., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattaset al., 1991 Mol. Cell. Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS86, 3519; Hatzoglou et al., 1991 J. Biol. Chem 266, 8416; Hatzoglou etal., 1988. J. Biol. Chem 263, 17798; Li et al., 1992 Hum. Gen. Ther. 3,381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol.Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al.,1994 Gene Ther 1,307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987PNAS 84, 5197). Thus, it is preferable to use an internal ribosome entrysite (IRES) to initiate translation of the second (and subsequent)coding sequence(s) in a poly-cistronic (or as used herein,“multi-cistronic”) message (Adam et al. 1991 J. Virol. 65, 4985).

Insertion of IRES elements into retroviral vectors is compatible withthe retroviral replication cycle and allows expression of multiplecoding regions from a single promoter (Adam et al. (as above); Koo etal. (1992) Virology 186:669-675; Chen et al. 1993 J. Virol67:2142-2148). IRES elements were first found in the non-translated 5′ends of picornaviruses where they promote cap-independent translation ofviral proteins (Jang et al. (1990) Enzyme 44; 292-309). When locatedbetween open reading frames in an RNA, IRES elements allow efficienttranslation of the downstream open reading frame by promoting entry ofthe ribosome at the IRES element followed by downstream initiation oftranslation.

The term “cistron” refers to a section of the DNA molecule thatspecifies the formation of one polypeptide chain, i.e. coding for onepolypeptide chain. For example, “bi-cistron” refers to two sections ofthe DNA molecule that specify the formation of two polypeptide chains,i.e. coding for two polypeptide chains; “tri-cistron” refers to threesections of the DNA molecule that specify the formation of threepolypeptide chains, i.e. coding for three polypeptide chains; etc. Theterm “multi-cistronic RNA” refers to an RNA that contains the geneticinformation to translate to several proteins. In contrast, amono-cistronic RNA contains the genetic information to translate only asingle protein. In the context of the present disclosure, themulti-cistronic RNA transcribed from the lentivirus may be translatedinto translated to two proteins, for example, a TCRα chain and TCRβchain.

A review on IRES is presented by Mountford and Smith (TIG May 1995 vol11, No 5:179-184). A number of different IRES sequences are knownincluding those from encephalomyocarditis virus (EMCV) (Ghattas, I. R.,et al., Mol. Cell. Biol., 11:5848-5859 (1991); BiP protein Macejak andSarnow, Nature 353:91 (1991); the Antennapedia gene of Drosophila (exonsd and e) Oh, et al., Genes & Development, 6:1643-1653 (1992) as well asthose in poliovirus (PV) Pelletier and Sonenberg, Nature 334: 320-325(1988); see also Mountford and Smith, TIG 11, 179-184 (1985).

According to WO-A-97/14809, IRES sequences are typically found in the 5′non-coding region of genes. In addition to those in the literature theycan be found empirically by looking for genetic sequences that affectexpression and then determining whether that sequence affects the DNA(i.e. acts as a promoter or enhancer) or only the RNA (acts as an IRESsequence).

IRES elements from PV, EMCV and swine vesicular disease virus havepreviously been used in retroviral vectors (Coffin et al, as above).

The term “IRES” includes any sequence or combination of sequences whichwork as or improve the function of an IRES. The IRES(s) may be of viralorigin (such as EMCV IRES, PV IRES, or FMDV 2A-like sequences) orcellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4IRES).

For the IRES to be capable of initiating translation of each NOI, itshould be located between or prior to NOIs in the vector genome. Forexample, for a multi-cistronic sequence containing in NOIs, the genomemay be as follows:

[NOI₁-IRES₁] . . . NOI_(n), n=any integer

For bi- and tri-cistronic sequences, the order may be as follows:

NOI₁-IRES₁-NOI₂

NOI₁-IRES₁-NOI₂-IRES₂-NOI₃

Alternative configurations of IRESs and NOIs can also be utilized. Forexample transcripts containing the IRESs and NOIs need not be drivenfrom the same promoter.

An example of this arrangement may be:

IRES₁-NOI₁-promoter-NOI₂-IRES₂-NOI₃.

In some aspects, in any construct utilizing an internal cassette havingmore than one IRES and NOI, the IRESs may be of different origins, thatis, heterologous to one another. For example, one IRES may be from EMCVand the other IRES may be from poliovirus.

Other Methods of Expressing Multiple Genes from One Vector

Although IRESs are an efficient way to co-express multiple genes fromone vector, other methods are also useful, and may be used alone or inconjunction with IRESs. These include the use of multiple internalpromoters in the vector (Overell et al., Mol Cell Biol. 8: 1803-8(1988)), or the use of alternate splicing patterns leading to multipleRNA species derived from the single viral genome that expresses thedifferent genes. This strategy has previously been used by itself fortwo genes (Cepko et al. Cell 37: 1053 (1984)).

For example, multiple cloning sites (MCS) can further be incorporatedinto the vector that facilitate the insertion of NOIs. This MCSfacilitates the introduction of any promoter, a single gene, two genesand optionally a gene inhibitory sequence, such as an antisense,ribozyme, shRNA, RNAi, microRNA, aptamer, transdominant mutant proteinor the like. A preferable embodiment is the expression of a gene ofinterest that has been modified so that its nucleotide sequence is codondegenerated with respect to the endogenous gene in a cell, andadditionally, the same vector expresses a gene inhibitory or silencingsequences capable of inhibiting or silencing the native gene ofinterest. This approach has enormous utility in the understanding thefunction of various protein domains by expressing the protein ofinterest that has been modified in these domains, and at the same timeexpressing a gene inhibitory or silencing sequence that represses orsilences expression of the native non-modified gene of interest. Thisapplication can also be used in gene therapeutic approaches for thetreatment of disease. For example, a lentiviral vector expressing anRNAi targeted to beta-hemoglobin can repress or silencesickle-hemoglobin in patients with sickle cell anemia. The samelentiviral vector can also express a normal hemoglobin molecule that hasbeen codon-degenerated at the site targeted by the RNAi. In this way,erythroid cells expressing sickle globin can repress sickle globinexpression, while expressing native hemoglobin and correct the geneticabnormality. The lentiviral vector could be delivered into a stem cellpopulation that would give rise to erythroid cells expressing hemoglobinthat would eventually become red cells. This approach can be used totreat a wide variety of diseases, including cancer, genetic disease andinfectious diseases.

Transduced Cells

The present invention also relates to a cell that has been transducedwith a vector system comprising a viral genome according to theinvention.

The cell may be transduced in vivo, in vitro or ex vivo by any suitablemeans. For example, if the cell is a cell from a mammalian subject, thecell may be removed from the subject and transduced ready forreimplantation into the subject (ex vivo transduction). Alternatively,the cell may be transduced by direct gene transfer in vivo, using thevector system of the present invention in accordance with standardtechniques (such as via injection of vector stocks expressing the NOIs).If the cell is part of a cell line that is stable in culture (i.e. whichcan survive numerous passages and can multiple in vitro) then it may betransduced in vitro by standard techniques, for example, by exposure ofthe cell to viral supernatants comprising vectors expressing the NOIs.

The cell may be any cell that is susceptible to transduction. If thevector system is capable of transducing non-dividing cells (for exampleif it is a lentiviral system) then the cell may be a non-dividing cell.

In one aspect, the present disclosure relates to activation,transduction, and/or expansion of immune cells, such as lymphocytes,neutrophils, and/or monocytes. In some aspects, the immune cells arelymphocytes, such as T cells (e.g., tumor-infiltrating lymphocytes, CD8+T cells, CD4+ T cells, and γδ T cells), B cells, and/or NK cells, thatmay be used for transgene expression. In another aspect, the disclosurerelates to activation, transduction, and expansion of γδ T cells whiledepleting α- and/or β-TCR positive cells.

In an aspect, whole PBMC population, without prior depletion of specificcell populations, such as monocytes, αβ T-cells, B-cells, and NK cells,can be activated and expanded. In another aspect, γδ T cells may beisolated from a complex sample that is cultured in vitro. In anotheraspect, enriched γδ T cell populations can be generated prior to theirspecific activation and expansion. In another aspect, activation andexpansion of T cells may be performed without the presence of native orengineered APCs. In another aspect, isolation and expansion of T cellsfrom tumor specimens can be performed using immobilized T cell mitogens,including antibodies specific to TCR, and other TCR activating agents,including lectins. In another aspect, isolation and expansion of T cellsfrom tumor specimens can be performed in the absence of T cell mitogens,including antibodies specific to TCR, and other TCR activating agents,including lectins.

In an aspect, T cells are isolated from leukapheresis of a subject, forexample, a human subject. In another aspect, T cells are not isolatedfrom peripheral blood mononuclear cells (PBMC).

T cell preparation may be performed by using methods disclosed inUS20190247433, the content of which is hereby incorporated by referencein its entirety.

In an aspect, the disclosure provides for methods of transducing a Tcell including thawing frozen PBMC, resting the thawed PBMC, activatingthe T cell in the cultured PBMC with an anti-CD3 antibody and ananti-CD28 antibody, transducing the activated T cell with a viralvector, expanding the transduced T cell, and obtaining the expanded Tcells.

In another aspect, the present disclosure relates to a method ofpreparing a T cell population, including obtaining fresh PBMC, i.e.,PBMC is not obtained by thawing cryopreserved PBMC, activating the Tcell in the fresh PBMC with an anti-CD3 antibody and an anti-CD28antibody, transducing the activated T cell with a viral vector,expanding the transduced T cell, and harvesting the expanded T cell.

In another embodiment of the present disclosure, for fresh PBMC, i.e.,not frozen, resting may not be needed. Thus, fresh PBMC, withoutresting, may be activated by anti-CD3 antibody and anti-CD28 antibody,followed by viral vector transduction to obtain transduced T cells.

In another aspect, the thawing, the resting, the activating, thetransducing, the expanding, and/or the obtaining may be performed in aclosed system.

In another aspect, the activating, the transducing, the expanding, andthe harvesting may be performed in a closed or semi-closed system.

In another aspect, the closed system may be CliniMACS, Prodigy™, WAVE(XURI™) Bioreactor, WAVE (XURI™) Bioreactor in combination with BioSafeSepax™ II, G-Rex/GatheRex™ closed system, or G-Rex/GatheRex™ closedsystem in combination with BioSafe Sepax™ II.

To produce T cells with improved efficacy for adoptive immunotherapy, Tcell may be prepared by using methods disclosed in US20190292520, thecontent of which is hereby incorporated by reference in its entirety.

In an aspect, methods for producing T cells with improved efficacy foradoptive immunotherapy may include obtaining T cells from at least onehealthy donor, patient, or individual, activating the T cells,transducing the activated T cells with a viral vector, expanding thetransduced T cells for about 3 days to about 5 days after activation,collecting the expanded transduced T cells for infusing into the atleast one healthy donor, patient, or individual, in which the efficacyfor adoptive immunotherapy of the T cells expanded for about 3 to about5 days is improved relative to activated and transduced T cells expandedfor about 7 days or more after activation.

In another aspect, the expanded T cells exhibit a naïve T cells (T_(N))and/or stem memory T cells (T_(scm))/T central memory (T_(cm))phenotype.

In another aspect, methods for producing T cells with improved efficacyfor adoptive immunotherapy may include obtaining a population of CD8+ Tcells from a patient or a donor, determining the percent of CD28+ CD8+ Tcells in the obtained population, activating the determined populationwith anti-CD3 antibody and anti-CD28 antibody, in which the determinedpopulation comprises at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99% of CD28+ CD8+ T cells, transducing theactivated T cell population with a viral vector, and expanding thetransduced T cell population.

In another aspect, the disclosure relates to ex vivo methods forproducing T cells with improved efficacy for immunotherapy including:determining in an isolated CD8+ T cell population a percent of CD28+CD8+ T cells, activating the determined population with anti-CD3antibody and anti-CD28 antibody, and provided that the determinedpopulation comprises at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of CD28+CD8+ T cells, transducing the activated T cell population with a viralvector, and expanding the transduced T cell population.

In another aspect, the disclosure relates to methods for producing Tcells with improved efficacy for immunotherapy including: obtaining apopulation of CD8+ T cells from a patient or a donor, determining thepercent of CD28+ CD8+ T cells in the obtained population, activating thedetermined TCR population with anti-CD3 antibody in the absence ofanti-CD28 antibody, provided that the determined population comprisesless than about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 9%, lessthan about 8%, less than about 7%, less than about 6%, less than about5%, less than about 4%, less than about 3%, less than about 2%, or lessthan about 1% of CD28+ CD8+ T cells, transducing the activated T cellpopulation with a viral vector, and expanding the transduced T cellpopulation.

In another aspect, the disclosure relates to ex vivo methods forproducing T cells with improved efficacy for immunotherapy including:determining in an isolated CD8+ T cell population the percent of CD28+CD8+ T cells, activating the determined TCR population with anti-CD3antibody in the absence of anti-CD28 antibody, provided that thedetermined population comprises less than about 50%, less than about45%, less than about 40%, less than about 35%, less than about 30%, lessthan about 25%, less than about 20%, less than about 15%, less thanabout 10%, less than about 9%, less than about 8%, less than about 7%,less than about 6%, less than about 5%, less than about 4%, less thanabout 3%, less than about 2%, or less than about 1% of CD28+ CD8+ Tcells, transducing the activated T cell population with a viral vector,and expanding the transduced T cell population.

In another aspect, the transducing and the expanding may be carried outin the presence of at least one cytokine.

In an aspect, the isolated γδ T cells can rapidly expand in response tocontact with one or more antigens. Some γδ T cells, such as Vγ9Vδ2+ Tcells, can rapidly expand in vitro in response to contact with someantigens, like prenyl-pyrophosphates, alkyl amines, and metabolites ormicrobial extracts during tissue culture. Stimulated γδ T-cells canexhibit numerous antigen-presentation, co-stimulation, and adhesionmolecules that can facilitate the isolation of γδ T-cells from a complexsample. γδ T cells within a complex sample can be stimulated in vitrowith at least one antigen for 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, or another suitable period of time. Stimulation of γδ Tcells with a suitable antigen can expand γδ T cell population in vitro.

Non-limiting examples of antigens that may be used to stimulate theexpansion of γδ T cells from a complex sample in vitro may include,prenyl-pyrophosphates, such as isopentenyl pyrophosphate (IPP),alkyl-amines, metabolites of human microbial pathogens, metabolites ofcommensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP),(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethylpyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallylphosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosinetriphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranylpyrophosphate (GGPP), isopentenyl-adenosine triphosphate (IPPPA),monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP),3-formyl-1-butyl-pyrophosphate (TUBAg 1), X-pyrophosphate (TUBAg 2),3-formyl-1-butyl-uridine triphosphate (TUBAg 3),3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg 4), monoethylalkylamines, allyl pyrophosphate, crotoyl pyrophosphate,dimethylallyl-γ-uridine triphosphate, crotoyl-γ-uridine triphosphate,allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine,iso-amylamine and nitrogen containing bisphosphonates.

Activation and expansion of γδ T cells can be performed using activationand co-stimulatory agents described herein to trigger specific γδ T cellproliferation and persistence populations. In an aspect, activation andexpansion of γδ T-cells from different cultures can achieve distinctclonal or mixed polyclonal population subsets. In another aspect,different agonist agents can be used to identify agents that providespecific γδ activating signals. In another aspect, agents that providespecific γδ activating signals can be different monoclonal antibodies(MAbs) directed against the γδ TCRs. In another aspect, companionco-stimulatory agents to assist in triggering specific γδ T cellproliferation without induction of cell energy and apoptosis can beused. These co-stimulatory agents can include ligands binding toreceptors expressed on γδ cells, such as NKG2D, CD161, CD70, JAML, DNAXaccessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM,CD122, DAP, and CD28. In another aspect, co-stimulatory agents can beantibodies specific to unique epitopes on CD2 and CD3 molecules. CD2 andCD3 can have different conformation structures when expressed on αβ orγδ T-cells. In another aspect, specific antibodies to CD3 and CD2 canlead to distinct activation of γδ T cells.

A population of γδ T-cells may be expanded ex vivo prior to engineeringof the γδ T-cells. Non-limiting example of reagents that can be used tofacilitate the expansion of a γδ T-cell population in vitro may includeanti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40antibodies, IL-2, IL-15, IL-12, IL-9, IL-33, IL-18, or IL-21, CD70 (CD27ligand), phytohaemagglutinin (PHA), concavalin A (ConA), pokeweed (PWM),protein peanut agglutinin (PNA), soybean agglutinin (SBA), Les CulinarisAgglutinin (LCA), Pisum Sativum Agglutinin (PSA), Helix pomatiaagglutinin (HPA), Vicia graminea Lectin (VGA), or another suitablemitogen capable of stimulating T-cell proliferation.

In an aspect, engineered (or transduced) γδ T cells can be expanded exvivo without stimulation by an antigen presenting cell oraminobisphosphonate. Antigen reactive engineered T cells of the presentdisclosure may be expanded ex vivo and in vivo. In another aspect, anactive population of engineered γδ T cells of the present disclosure maybe expanded ex vivo without antigen stimulation by an antigen presentingcell, an antigenic peptide, a non-peptide molecule, or a small moleculecompound, such as an aminobisphosphonate but using certain antibodies,cytokines, mitogens, or fusion proteins, such as IL-17 Fc fusion, MICAFc fusion, and CD70 Fc fusion. Examples of antibodies that can be usedin the expansion of a γδ T-cell population include anti-CD3, anti-CD27,anti-CD30, anti-CD70, anti-OX40, anti-NKG2D, or anti-CD2 antibodies,examples of cytokines may include IL-2, IL-15, IL-12, IL-21, IL-18,IL-9, IL-7, and/or IL-33, and examples of mitogens may include CD70 theligand for human CD27, phytohaemagglutinin (PHA), concavalin A (ConA),pokeweed mitogen (PWM), protein peanut agglutinin (PNA), soybeanagglutinin (SBA), les culinaris agglutinin (LCA), Pisum sativumagglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea Lectin(VGA) or another suitable mitogen capable of stimulating T-cellproliferation. In another aspect, a population of engineered γδ T cellscan be expanded in less than 60 days, less than 48 days, less than 36days, less than 24 days, less than 12 days, or less than 6 days. Inanother aspect, a population of engineered γδ T cells can be expandedfrom about 7 days to about 49 days, about 7 days to about 42 days, fromabout 7 days to about 35 days, from about 7 days to about 28 days, fromabout 7 days to about 21 days, or from about 7 days to about 14 days.

In another aspect, the present disclosure provides methods for the exvivo expansion of a population of engineered T-cells for adoptivetransfer therapy. Engineered T cells of the disclosure may be expandedex vivo. Engineered T cells of the disclosure can be expanded in vitrowithout activation by APCs, or without co-culture with APCs, andaminophosphates.

The ability of T cells to recognize a broad spectrum of antigens can beenhanced by genetic engineering of the T cells. In an aspect, T cellscan be engineered to provide a universal allogeneic therapy thatrecognizes an antigen of choice in vivo. Genetic engineering of theT-cells may include stably integrating a construct expressing a tumorrecognition moiety, such as αβ TCR, γδ TCR, chimeric antigen receptor(CAR), which combines both antigen-binding and T-cell activatingfunctions into a single receptor, an antigen binding fragment thereof,or a lymphocyte activation domain into the genome of the isolatedT-cell(s), a cytokine (for example, IL-15, IL-12, IL-2. IL-7. IL-21,IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL1β) to enhance T-cellproliferation, survival, and function ex vivo and in vivo. Geneticengineering of the isolated T-cell may also include deleting ordisrupting gene expression from one or more endogenous genes in thegenome of the isolated T-cells, such as the MHC locus (loci).

Chimeric Antigen Receptors (CARs)

Embodiments of the present disclosure may include introducing nucleicacids that encode one or more CARs into T cells. T cells may be αβ Tcells, γδ T cells, or natural killer T cells. In various embodiments,the present disclosure provides T cells genetically engineered withvectors designed to express CARs that redirect cytotoxicity toward tumorcells. CARs are molecules that combine antibody-based specificity for atarget antigen, e.g., tumor antigen, with a T cell receptor-activatingintracellular domain to generate a chimeric protein that exhibits aspecific anti-tumor cellular immune activity. As used herein, the term,“chimeric,” describes being composed of parts of different proteins orDNAs from different origins.

CARs may contain an extracellular domain that binds to a specific targetantigen (also referred to as a binding domain or antigen-specificbinding domain), a transmembrane domain and an intracellular signalingdomain. The main characteristic of CARs may be their ability to redirectimmune effector cell specificity, thereby triggering proliferation,cytokine production, phagocytosis or production of molecules that canmediate cell death of the target antigen expressing cell in a majorhistocompatibility (MHC) independent manner, exploiting the cellspecific targeting abilities of monoclonal antibodies, soluble ligandsor cell specific coreceptors.

In particular embodiments, CARs may contain an extracellular bindingdomain including but not limited to an antibody or antigen bindingfragment thereof, a tethered ligand, or the extracellular domain of acoreceptor, that specifically binds a target antigen that is atumor-associated antigen (TAA) or a tumor-specific antigen (TSA). Incertain embodiments, the TAA or TSA may be expressed on a blood cancercell. In another embodiment, the TAA or TSA may be expressed on a cellof a solid tumor. In particular embodiments, the solid tumor may be aglioblastoma, a non-small cell lung cancer, a lung cancer other than anon-small cell lung cancer, breast cancer, prostate cancer, pancreaticcancer, liver cancer, colon cancer, stomach cancer, a cancer of thespleen, skin cancer, a brain cancer other than a glioblastoma, a kidneycancer, a thyroid cancer, or the like.

In particular embodiments, the TAA or TSA may be selected from the groupconsisting of alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3,B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70,CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR familyincluding ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP,fetal AchR, FRα, GD2, GD3, *Glypican-3 (GPC3), HLA-A1+MAGE1,HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin,Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1,SSX, Survivin, TAG72, TEMs, and VEGFR2.

Binding Domains of CARs

In particular embodiments, CARs contemplated herein comprise anextracellular binding domain that specifically binds to a targetpolypeptide, e.g., target antigen, expressed on tumor cell. As usedherein, the terms, “binding domain,” “extracellular domain,”

“extracellular binding domain,” “antigen-specific binding domain,” and“extracellular antigen specific binding domain,” may be usedinterchangeably and provide a CAR with the ability to specifically bindto the target antigen of interest. A binding domain may include anyprotein, polypeptide, oligopeptide, or peptide that possesses theability to specifically recognize and bind to a biological molecule(e.g., a cell surface receptor or tumor protein, lipid, polysaccharide,or other cell surface target molecule, or component thereof). A bindingdomain may include any naturally occurring, synthetic, semi-synthetic,or recombinantly produced binding partner for a biological molecule ofinterest.

In particular embodiments, the extracellular binding domain of a CAR mayinclude an antibody or antigen binding fragment thereof. An “antibody”refers to a binding agent that is a polypeptide containing at least alight chain or heavy chain immunoglobulin variable region, whichspecifically recognizes and binds an epitope of a target antigen, suchas a peptide, lipid, polysaccharide, or nucleic acid containing anantigenic determinant, such as those recognized by an immune cell.Antibodies may include antigen binding fragments thereof. The term mayalso include genetically engineered forms, such as chimeric antibodies(for example, humanized murine antibodies), hetero-conjugate antibodies,e.g., bispecific antibodies, and antigen binding fragments thereof. Seealso, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., NewYork, 1997.

In particular embodiments, the target antigen may be an epitope of analpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX,CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a,CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family includingErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetalAchR, FRα, GD2, GD3, *Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1,HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1,IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16,NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin,TAG72, TEMs, or VEGFR2 polypeptide.

Light and heavy chain variable regions may contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The CDRs can be definedor identified by conventional methods, such as by sequence according toKabat et al (Wu, T T and Kabat, E. A., J Exp Med. 132(2):211-50, (1970);Borden, P. and Kabat E. A., PNAS, 84: 2440-2443 (1987); (see, Kabat etal, Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference), or by structure according to Chothia et al (Choithia, C. andLesk, A. M., J Mol. Biol, 196(4): 901-917 (1987), Choithia, C. et al,Nature, 342: 877-883 (1989)). The contents of the afore-mentionedreferences are hereby incorporated by reference in their entireties. Thesequences of the framework regions of different light or heavy chainsmay be relatively conserved within a species, such as humans. Theframework region of an antibody that is the combined framework regionsof the constituent light and heavy chains may serve to position andalign the CDRs in three-dimensional space. The CDRs may be primarilyresponsible for binding to an epitope of an antigen. The CDRs of eachchain may be typically referred to as CDR1, CDR2, and CDR3, numberedsequentially starting from the N-terminus, and may be also typicallyidentified by the chain, in which the particular CDR is located. Thus,the CDRs located in the variable domain of the heavy chain of theantibody may be referred to as CDRH1, CDRH2, and CDRH3, whereas the CDRslocated in the variable domain of the light chain of the antibody arereferred to as CDRL1, CDRL2, and CDRL3. Antibodies with differentspecificities (i.e., different combining sites for different antigens)may have different CDRs. Although it is the CDRs that vary from antibodyto antibody, only a limited number of amino acid positions within theCDRs are directly involved in antigen binding. These positions withinthe CDRs are called specificity determining residues (SDRs).

References to “VH” or “VH” refers to the variable region of animmunoglobulin heavy chain, including that of an antibody, Fv, scFv,dsFv, Fab, or other antibody fragment. References to “VL” or “VL” refersto the variable region of an immunoglobulin light chain, including thatof an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes or by a cell into which the light and heavy chain genes of asingle antibody have been transfected. Monoclonal antibodies may beproduced by methods known to those of skill in the art, for example, bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies may include humanizedmonoclonal antibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a mouse. In particular preferred embodiments, a CARdisclosed herein may contain antigen-specific binding domain that is achimeric antibody or antigen binding fragment thereof.

In certain embodiments, the antibody may be a humanized antibody (suchas a humanized monoclonal antibody) that specifically binds to a surfaceprotein on a tumor cell. A “humanized” antibody is an immunoglobulinincluding a human framework region and one or more CDRs from a non-human(for example a mouse, rat, or synthetic) immunoglobulin. Humanizedantibodies can be constructed by means of genetic engineering (see forexample, U.S. Pat. No. 5,585,089, the content of which is herebyincorporated by reference in its entirety).

In embodiments, the extracellular binding domain of a CAR may contain anantibody or antigen binding fragment thereof, including but not limitedto a Camel Ig (a camelid antibody (VHH)), Ig NAR, Fab fragments, Fab′fragments, F(ab)′2 fragments, F(ab)′3 fragments, Fv, single chain Fvantibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody,tetrabody, disulfide stabilized Fv protein (“dsFv”), and single-domainantibody (sdAb, Nanobody).

“Camel Ig” or “camelid VHH” as used herein refers to the smallest knownantigen-binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEBJ., 21:3490-3498 (2007), the content of which is hereby incorporated byreference in its entirety). A “heavy chain antibody” or a “camelidantibody” refers to an antibody that contains two VH domains and nolight chains (Riechmann L. et al, J. Immunol. Methods 231:25-38 (1999);WO94/04678; WO94/25591; U.S. Pat. No. 6,005,079; the contents of whichare hereby incorporated by reference in its entirety).

“IgNAR” of “immunoglobulin new antigen receptor” refers to class ofantibodies from the shark immune repertoire that consist of homodimersof one variable new antigen receptor (VNAR) domain and five constant newantigen receptor (CNAR) domains.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. The Fab fragment contains the heavy- andlight-chain variable domains and also contains the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxy terminus of the heavy chain CH1 domain includingone or more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)2 antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In a single-chain Fv (scFv) species, one heavy-and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al, Nat. Med. 9:129-134 (2003); andHollinger et al, PNAS USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al, Nat. Med. 9:129-134(2003). The contents of the afore-mentioned references are herebyincorporated by reference in their entireties.

“Single domain antibody” or “sdAb” or “nanobody” refers to an antibodyfragment that consists of the variable region of an antibody heavy chain(VH domain) or the variable region of an antibody light chain (VLdomain) (Holt, L., et al, Trends in Biotechnology, 21(11): 484-490, thecontent of which is hereby incorporated by reference in its entirety).

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain and in either orientation {e.g., VL-VH or VH-VL).Generally, the scFv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains which enables the scFv to form the desiredstructure for antigen binding. For a review of scFv, see, e.g.,Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315, the content of which is hereby incorporated by reference in itsentirety.

In a certain embodiment, the scFv binds an alpha folate receptor, 5T4,αvβ6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20, CD22, CD30, CD33,CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA,CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40,EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *Glypican-3 (GPC3),HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1,HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y,Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME,PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, or VEGFR2 polypeptide.

Linkers of CARs

In certain embodiments, the CARs may contain linker residues between thevarious domains, e.g., between VH and VL domains, added for appropriatespacing and conformation of the molecule. CARs may contain one, two,three, four, or five or more linkers. In particular embodiments, thelength of a linker may be about 1 to about 25 amino acids, about 5 toabout 20 amino acids, or about 10 to about 20 amino acids, or anyintervening length of amino acids. In some embodiments, the linker maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, or more amino acids long. Illustrative examplesof linkers include glycine polymers (G)n; glycine-serine polymers(Gi_sSi_5)n, where n is an integer of at least one, two, three, four, orfive; glycine-alanine polymers; alanine-serine polymers; and otherflexible linkers known in the art. Glycine and glycine-serine polymersare relatively unstructured, and therefore may be able to serve as aneutral tether between domains of fusion proteins, such as CARs. Glycinemay access significantly more phi-psi space than even alanine, and maybe much less restricted than residues with longer side chains (seeScheraga, Rev. Computational Chem. 11173-142 (1992), the content ofwhich is hereby incorporated by reference in its entirety). Theordinarily skilled artisan may recognize that design of a CAR inparticular embodiments can include linkers that may be all or partiallyflexible, such that the linker can include a flexible linker as well asone or more portions that confer less flexible structure to provide fora desired CAR structure.

In particular embodiments a CAR may include a scFV that may furthercontain a variable region linking sequence. A “variable region linkingsequence,” is an amino acid sequence that connects a heavy chainvariable region to a light chain variable region and provides a spacerfunction compatible with interaction of the two sub-binding domains sothat the resulting polypeptide retains a specific binding affinity tothe same target molecule as an antibody that may contain the same lightand heavy chain variable regions. In one embodiment, the variable regionlinking sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids long. Ina particular embodiment, the variable region linking sequence maycontain a glycine-serine polymer (Gi_sSi_5)n, where n is an integer ofat least 1, 2, 3, 4, or 5. In another embodiment, the variable regionlinking sequence comprises a (G₄S)₃ amino acid linker.

Spacer Domains of CARs

In particular embodiments, the binding domain of the CAR may be followedby one or more “spacer domains,” which refers to the region that movesthe antigen binding domain away from the effector cell surface to enableproper cell/cell contact, antigen binding and activation (Patel et al,Gene Therapy, 1999; 6: 412-419, the content of which is herebyincorporated by reference in its entirety). The spacer domain may bederived either from a natural, synthetic, semi-synthetic, or recombinantsource. In certain embodiments, a spacer domain may be a portion of animmunoglobulin, including, but not limited to, one or more heavy chainconstant regions, e.g., CH2 and CH3. The spacer domain can include theamino acid sequence of a naturally occurring immunoglobulin hinge regionor an altered immunoglobulin hinge region. In one embodiment, the spacerdomain may include the CH2 and CH3 of IgG1.

Hinge Domains of CARs

The binding domain of CAR may be generally followed by one or more“hinge domains,” which may play a role in positioning the antigenbinding domain away from the effector cell surface to enable propercell/cell contact, antigen binding and activation. CAR generally mayinclude one or more hinge domains between the binding domain and thetransmembrane domain (TM). The hinge domain may be derived either from anatural, synthetic, semi-synthetic, or recombinant source. The hingedomain can include the amino acid sequence of a naturally occurringimmunoglobulin hinge region or an altered immunoglobulin hinge region.Illustrative hinge domains suitable for use in the CARs may include thehinge region derived from the extracellular regions of type 1 membraneproteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type hingeregions from these molecules or may be altered. In another embodiment,the hinge domain may include a CD8a hinge region.

Transmembrane (TM) Domains of CARs

The “transmembrane domain” may be the portion of CAR that can fuse theextracellular binding portion and intracellular signaling domain andanchors CAR to the plasma membrane of the immune effector cell. The TMdomain may be derived either from a natural, synthetic, semi-synthetic,or recombinant source. Illustrative TM domains may be derived from(including at least the transmembrane region(s) of) the α, β, or ζ chainof the T-cell receptor, CD3ε, CD3∂, CD4, CD5, CD9, CD16, CD22, CD27,CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, and CD154. Inone embodiment, CARs may contain a TM domain derived from CD8a. Inanother embodiment, a CAR contemplated herein comprises a TM domainderived from CD8a and a short oligo- or polypeptide linker, preferablybetween 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length thatlinks the TM domain and the intracellular signaling domain of CAR. Aglycine-serine linker provides a particularly suitable linker.

Intracellular Signaling Domains of CARs

In particular embodiments, CARs may contain an intracellular signalingdomain. An “intracellular signaling domain,” refers to the part of a CARthat participates in transducing the message of effective CAR binding toa target antigen into the interior of the immune effector cell to eliciteffector cell function, e.g., activation, cytokine production,proliferation and cytotoxic activity, including the release of cytotoxicfactors to the CAR-bound target cell, or other cellular responseselicited with antigen binding to the extracellular CAR domain.

The term “effector function” refers to a specialized function of thecell. Effector function of the T cell, for example, may be cytolyticactivity or help or activity including the secretion of a cytokine.Thus, the term “intracellular signaling domain” refers to the portion ofa protein, which can transduce the effector function signal and thatdirect the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain may be used, suchtruncated portion may be used in place of the entire domain as long asit can transduce the effector function signal. The term intracellularsignaling domain may be meant to include any truncated portion of theintracellular signaling domain sufficient to transducing effectorfunction signal.

It is known that signals generated through TCR alone are insufficientfor full activation of the T cell and that a secondary or costimulatorysignal may be also required. Thus, T cell activation can be said to bemediated by two distinct classes of intracellular signaling domains:primary signaling domains that initiate antigen-dependent primaryactivation through the TCR (e.g., a TCR/CD3 complex) and costimulatorysignaling domains that act in an antigen-independent manner to provide asecondary or costimulatory signal. In preferred embodiments, CAR mayinclude an intracellular signaling domain that may contain one or more“costimulatory signaling domain” and a “primary signaling domain.”Primary signaling domains can regulate primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primarysignaling domains that act in a stimulatory manner may contain signalingmotifs, which are known as immunoreceptor tyrosine-based activationmotifs or ITAMs. Illustrative examples of ITAM containing primarysignaling domains that are of particular use in the invention mayinclude those derived from TCRζ, FcRγ. FcRβ, CD3γ, CD3δ, CD3ε, CD3ζCD22, CD79a, CD79b, and CD66d. In particular preferred embodiments, CARmay include a CD3ζ primary signaling domain and one or morecostimulatory signaling domains. The intracellular primary signaling andcostimulatory signaling domains may be linked in any order in tandem tothe carboxyl terminus of the transmembrane domain.

CARs may contain one or more costimulatory signaling domains to enhancethe efficacy and expansion of T cells expressing CAR receptors. As usedherein, the term, “costimulatory signaling domain,” or “costimulatorydomain”, refers to an intracellular signaling domain of a costimulatorymolecule. Illustrative examples of such costimulatory molecules mayinclude CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS(CD278), CTLA4, LFA-1, CD2, CD7, LIGHT, TRIM, LCK3, SLAM, DAP10, LAG3,HVEM and NKD2C, and CD83. In one embodiment, CAR may contain one or morecostimulatory signaling domains selected from the group consisting ofCD28, CD137, and CD134, and a CD3ζ primary signaling domain.

In one embodiment, CAR may contain an scFv that binds an alpha folatereceptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20,CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123,CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2),EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2,GD3, *Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+M AGE1, HLA-A3+MAGE1,HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2,Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands,NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, or VEGFR2polypeptide; a transmembrane domain derived from a polypeptide selectedfrom the group consisting of: CD8α; CD4, CD45, PD1, and CD152; and oneor more intracellular costimulatory signaling domains selected from thegroup consisting of: CD28, CD54, CD134, CD137, CD152, CD273, CD274, andCD278; and a CD3ζ primary signaling domain.

In another embodiment, CAR may contain an scFv that binds an alphafolate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19,CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b,CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2(HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR,FRα, GD2, GD3, *Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1,HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1,IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16,NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin,TAG72, TEMs, or VEGFR2 polypeptide; a hinge domain selected from thegroup consisting of: IgG1 hinge/CH2/CH3 and CD8α, and CD8α; atransmembrane domain derived from a polypeptide selected from the groupconsisting of: CD8α; CD4, CD45, PD1, and CD152; and one or moreintracellular costimulatory signaling domains selected from the groupconsisting of: CD28, CD 134, and CD 137; and a CD3ζ primary signalingdomain.

In yet another embodiment, CAR may contain an scFv, further including alinker, that binds an alpha folate receptor, 5T4, αvβ6 integrin, BCMA,B7-H3, B7-H6, CAIX, CD 19, CD20, CD22, CD30, CD33, CD44, CD44v6,CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR,EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2,EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *Glypican-3 (GPC3), HLA-A1+MAGE1,HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1,HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin,Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1,SSX, Survivin, TAG72, TEMs, or VEGFR2 polypeptide; a hinge domainselected from the group consisting of: IgG1 hinge/CH2/CH3 and CD8α, andCD8α; a transmembrane domain comprising a TM domain derived from apolypeptide selected from the group consisting of: CD8α; CD4, CD45, PD1,and CD 152, and a short oligo- or polypeptide linker, preferably between1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TMdomain to the intracellular signaling domain of the CAR; and one or moreintracellular costimulatory signaling domains selected from the groupconsisting of: CD28, CD 134, and CD137; and a CD3ζ primary signalingdomain.

In a particular embodiment, CAR may contain an scFv that binds an alphafolate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19,CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b,CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2(HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR,FRα, GD2, GD3, *Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+M AGE1,HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1,IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16,NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin,TAG72, TEMs, or VEGFR2 polypeptide; a hinge domain containing a CD8apolypeptide; a CD8a transmembrane domain containing a polypeptide linkerof about 3 amino acids; one or more intracellular costimulatorysignaling domains selected from the group consisting of: CD28, CD134,and CD137; and a CD3ζ primary signaling domain.

Engineered T-cells may be generated with various methods. For example, apolynucleotide encoding an expression cassette that comprises a tumorrecognition, or another type of recognition moiety, can be stablyintroduced into the T-cell by a transposon/transposase system or aviral-based gene transfer system, such as a lentiviral or a retroviralsystem, or another suitable method, such as transfection,electroporation, transduction, lipofection, calcium phosphate (CaPO₄),nanoengineered substances, such as Ormosil, viral delivery methods,including adenoviruses, retroviruses, lentiviruses, adeno-associatedviruses, or another suitable method. A number of viral methods have beenused for human gene therapy, such as the methods described in WO1993020221, the content of which is incorporated herein in its entirety.Non-limiting examples of viral methods that can be used to engineer Tcells may include γ-retroviral, adenoviral, lentiviral, herpes simplexvirus, vaccinia virus, pox virus, or adeno-virus associated viralmethods.

In an aspect, constructs and vectors described herein are used with themethodology described in U.S. Ser. No. 16/200,308, filed on Nov. 26,2018, the contents of which are incorporated by reference in theirentirety.

Cassettes

The present invention can employ cassettes comprising one or more NOIs,which, in the case of two or more NOIs, can be operably linked by anIRES. These cassettes may be used in a method for producing the vectorgenome in a producer cell.

The present invention also provides an expression vector comprising sucha cassette. Transfection of a suitable cell with such an expressionvector should result in a cell that expresses each POI encoded by theNOI in the cassette. The present invention also provides such atransfected cell.

Cloning of the cassette into an expression vector and transfection ofcells with the vector (to give expression of the cassette) can becarried out by techniques well known in the art (such as those describedin Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory Press (1989)), and other laboratorytextbooks).

In some aspects, the cassette comprises a promoter.

In some aspects, the cassette comprises one NOI. The NOI can be any NOIas described in detail above. For example, the NOI may have atherapeutic or diagnostic application. Suitable NOIs include, but arenot limited to: sequences encoding enzymes, cytokines, chemokines,hormones, antibodies, anti-oxidant molecules, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,immune co-stimulatory molecules, immunomodulatory molecules, anti-senseRNA, small interfering RNA (siRNA), a trans dominant negative mutant ofa target protein, a toxin, a conditional toxin, an antigen, an antigenreceptor, a chimeric antigen receptor, a T-cell receptor, a tumorsuppressor protein, and growth factors, membrane proteins, pro- andanti-angiogenic proteins and peptides, vasoactive proteins and peptides,antiviral proteins and ribozymes, and derivatives thereof (such as withan associated reporter group).

In some aspects, the cassette comprises two or more NOIs. A cassettecomprising two or more NOIs can be bi-cistronic or tri-cistronic, andcan comprise the following elements:

Promoter-(NOI₁)-(IRES₁)-(NOI₂)

Promoter-(NOI₁)-(IRES₁)-(NOI₂)-(IRES₂)-(NOI₃)

In some embodiments, a single lentiviral cassette can be used to createa single lentiviral vector, expressing one or more proteins. Inparticular, a single lentiviral cassette can be used to create a singlelentiviral vector expressing at least four individual monomer proteinsof two distinct dimers from a single multi-cistronic mRNA so as toco-express the dimers on the cell surface. For example, the integrationof a single copy of the lentiviral vector has been shown to besufficient to transform γδ T cells to co-express TCRαβ and CD8αβ.

In one aspect, the present disclosure relates to vectors containing amulti-cistronic cassette within a single vector capable of expressingmore than one, more than two, more than three, more than four genes,more than five genes, or more than six genes, in which the polypeptidesencoded by these genes may interact with one another, or may formdimers. The dimers may be homodimers, i.e., two identical proteinsforming a dimer, or heterodimers, i.e., two structurally differentproteins forming a dimer.

In one aspect, a lentiviral vector may contain a first nucleotidesequence S1 encoding a protein Z1, a second nucleotide sequence S2encoding a protein Z2, a third nucleotide sequence S3 encoding a proteinY1, and a fourth nucleotide sequence S4 encoding a protein Y2, in whichZ1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, in whichthe first dimer Z1Z2 is different from the second dimer Y1Y2.

In one aspect, a first lentiviral vector may contain a bi-cistroniccassette (2-in-1) encoding a dimer Z1Z2, and a second lentiviral vectormay contain a bi-cistronic cassette (2-in-1) encoding a dimer Y1Y2. Inthe 2-in-1 vectors, S1 and S2 may be arranged in tandem in a 5′ to 3′orientation of S1-S2 or S2-S1. Likewise, in the 2-in-1 vectors, S3 andS4 may be arranged in tandem in a 5′ to 3′ orientation of S3-S4 orS4-S3. Z1 and Z2 or Y1 and Y2 may be separated by one or moreself-cleaving 2A peptides.

In another aspect, a single lentiviral vector (4-in-1) may encode bothdistinct dimers Z1Z2 and Y1Y2, in which Z1, Z2, Y1, and Y2 may beseparated by one or more self-cleaving 2A peptides. For example, the S1,S2, S3, and S4 may be arranged in tandem in a 5′ to 3′ orientationselected from S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2,S1-S4-S3-S2, S1-S4-S2-S3, S2-S1-S3-S4, S2-S1-S4-S3, S2-S3-S1-S4,S2-S3-S4-S1, S2-S4-S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S3-S1-S4-S2,S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3-S4-S2-S1, S4-S1-S2-S3,S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2, or S4-S3-S2-S1.

In an aspect, the dimer Z1Z2 and/or the dimer Y1Y2 may be TCRs having aTCRα chain and a TCR chain or TCRs having a TCRγ chain and a TCRδ chain.

In an aspect, TCRs and antigen binding proteins that are capable of usewith the constructs, methods and embodiments described herein include,for example, those listed in Table 3 (SEQ ID NOs: 13-92) and those TCRsand antigen binding proteins described in U.S. Publication 20170267738,U.S. Publication 20170312350, U.S. Publication 20180051080, U.S.Publication 20180164315, U.S. Publication 20180161396, U.S. Publication20180162922, U.S. Publication 20180273602, U.S. Publication 20190016801,U.S. Publication 20190002556, and U.S. Publication 20190135914, thecontents of each of these publications and sequence listings describedtherein are herein incorporated by reference in their entireties.

In an aspect, TCRs and antigen binding proteins that are capable of usewith the constructs, methods and embodiments described herein include,for example, TCRs and antigen binding proteins that bind to binds to a“target antigenic (TA) peptide”.

The “target antigenic (TA) peptide” as used in context of the presentinvention refers to peptides which have been isolated and identifiedfrom infected or tumorous material, such as material isolated fromindividuals suffering from tuberculosis, or from an infection of theEpstein-Barr virus or from cancer. The protein from which the TA peptideis derived is subject to antigen processing in an infected cell or atumor cell, ten presented at the cell surface by the MHC molecule andthe cell, in particular the TA peptide/MHC complex can thus berecognized by immune effector cells of the host, such as T-cells or NKTcells. The TA peptide in context of the present invention comprises orconsists of 10, 12 or 14, such as 8 to 14, 8 to 12, for example 9 to 11amino acids. In context of the present invention, when it is referred toa specific TA peptide, it is referred to TA-C. Examples of TA antigenicpeptides, such as TA-C peptides are viral antigenic peptides, bacterialantigenic peptides or tumour associated antigen (TAA) antigenicpeptides, preferably TAA antigenic peptides. Accordingly, in oneembodiment, the TA antigenic peptide, in particular the TA-C, is a viralpeptide, a bacterial peptide or a tumour associated antigen (TAA)antigenic peptide, preferably a TAA antigenic peptide.

A “viral antigenic peptide” in context of the present invention is anantigenic peptide that is presented by the MHC molecule on the surfaceof a diseased cell and is of a viral origin, i.e. the cell is typicallyinfected by said virus. Such viral antigenic peptides have beendiscovered in context of infections from, for example, humanimmunodeficiency viruses (HIV), Human Cytomegalovirus (HCMV),cytomegalovirus (CMV), human papillomavirus (HPV), Hepatitis B virus(HBV), Hepatitis C virus (HCV), human papillomavirus infection (HPV),Epstein-Barr virus (EBV), Influenza virus. Accordingly, the viralantigenic peptide in context of the present invention may be anantigenic peptide selected from the group consisting of HIV antigenicpeptides, HCMV antigenic peptide, CMV antigenic peptides, HPV antigenicpeptides, HBV antigenic peptides; HCV antigenic peptides; EBV antigenicpeptides, Influenza antigenic peptides, preferably HIV, HBV, Influenzaand HCMV antigenic peptides.

Viral antigenic peptides that are capable of use with methods andembodiments described herein include, for example, those viral antigenicpeptides described in in the table herein below. In an aspect, viralantigenic peptides that are capable of use with the methods andembodiments described herein include at least one viral antigenicpeptide comprising or consisting of an amino acid sequence selected fromthe amino acid sequences of SEQ ID NO: 96 to SEQ ID NO: 98, as depictedherein below in table 1.

TABLE 1 List of viral antigenic peptides SEQ ID NO: Peptide Virus MHC 96SLYNTVATL HIV HLA-A*02:01 97 GILGFVFTL Influenza A HLA-A*02:01 98NLVPMVATV HCMV HLA-A*02:01

A “bacterial antigenic peptide” in context of the present invention isan antigenic peptide that is presented by the MHC molecule on thesurface of a diseased cell and is of a bacterial origin, i.e. the cellis typically infected by said bacteria. Such bacterial antigenicpeptides have been discovered in context of infections from, forexample, Mycobacterium tuberculosis. Accordingly, the bacterialantigenic peptide in context of the present invention may be aMycobacterium tuberculosis antigenic peptide.

“Tumor-associated antigens (TAA) peptides” also referred to as “TAApeptides” herein denotes peptides which have been isolated andidentified from tumorous material and which underwent antigen processingin a tumor cell and can thus be recognized by immune effector cells ofthe host. The TAA peptides comprises or consists of 10, 12 or 14, suchas 8 to 14, 8 to 12, for example 9 to 11 amino acids. The TAA peptidesin context of the present invention may be for example a cancer/testis(CT) antigenic peptide. Examples of cancer/testis (CT) antigenicpeptides are the MAGE-A antigenic peptide of the amino acid sequence ofSEQ ID NO: 216 and the PRAME antigenic peptide of the amino acidsequence of SEQ ID NO: 148. The TAA peptide in context of the presentinvention comprises a T-cell epitope and may also be referred to as TAApeptide, in a general context, and as TAA peptide C in context of thepresent invention when it is referred to one specific TAA peptide.

In an aspect, tumor associated antigen (TAA) peptides that are capableof use with methods and embodiments described herein include, forexample, those TAA peptides described in U.S. Publication 20160187351,U.S. Publication 20170165335, U.S. Publication 20170035807, U.S.Publication 20160280759, U.S. Publication 20160287687, U.S. Publication20160346371, U.S. Publication 20160368965, U.S. Publication 20170022251,U.S. Publication 20170002055, U.S. Publication 20170029486, U.S.Publication 20170037089, U.S. Publication 20170136108, U.S. Publication20170101473, U.S. Publication 20170096461, U.S. Publication 20170165337,U.S. Publication 20170189505, U.S. Publication 20170173132, U.S.Publication 20170296640, U.S. Publication 20170253633, U.S. Publication20170260249, U.S. Publication 20180051080, and U.S. Publication No.20180164315, the contents of each of these publications and sequencelistings described therein are herein incorporated by reference in theirentireties.

In an aspect, the bispecific antigen binding proteins described herein,in particular the antigen binding site B in context of the presentinvention, selectively recognize cells which present a TAA peptidedescribed in one of more of the patents and publications describedabove. In another aspect, TAA that are capable of use with the methodsand embodiments described herein include at least one TAA consisting ofan amino acid sequence selected from the amino acid sequences of SEQ IDNO: 99 to 256, preferably SEQ ID NO: 148 and 216. In an aspect, thebispecific antigen binding proteins, in particular the antigen bindingsite B of the bispecific antigen binding proteins, selectively recognizecells which present a TAA peptide/MHC complex, wherein the TAA peptidecomprises or consist of an amino acid sequence of SEQ ID NO: 99 to 256,or any of the amino acid sequences described in the patents orapplications described herein, preferably SEQ ID NO: 148 and 216.

Furthermore, the TA antigenic peptide in context of the presentinvention is a specific ligand of MHC-class-1-molecules orMHC-class-II-molecules, preferably MHC-class-I-molecules.

In context of the present invention, the TAA antigenic peptide C ispreferably selected from the group of TAA antigenic peptides consistingof the amino acids sequence of SEQ ID NO: 99 to 256, preferably thePRAME antigenic peptide comprising or consisting of the amino acidsequence ‘SLLQHLIGL’ of SEQ ID NO: 148 or the MAGE-A antigenic peptidecomprising or consisting of the amino acid sequence ‘KVLEHVVRV’ of SEQID NO: 216, more preferably SEQ ID NO: 216, wherein the MHC ispreferably a HLA-A*02.

In another aspect, the dimer Z1Z2 and/or the dimer Y1Y2 may be TCRαchain and TCRβ chain selected from R11KEA (SEQ ID NO: 13 and 14),R20P1H7 (SEQ ID NO: 15 and 16), R7P1D5 (SEQ ID NO: 17 and 18), R10P2G12(SEQ ID NO: 19 and 20), R10P1A7 (SEQ ID NO: 21 and 22), R4P1D10 (SEQ IDNO: 23 and 24), R4P3F9 (SEQ ID NO: 25 and 26), R4P3F9-B4 (SEQ ID NO: 25and 92), R4P3F9-A1B4 (SEQ ID NO: 91 and 92), R4P3H3 (SEQ ID NO: 27 and28), R36P3F9 (SEQ ID NO: 29 and 30), R52P2G11 (SEQ ID NO: 31 and 32),R53P2A9 (SEQ ID NO: 33 and 34), R26P1A9 (SEQ ID NO: 35 and 36), R26P2A6(SEQ ID NO: 37 and 38), R26P3H1 (SEQ ID NO: 39 and 40), R35P3A4 (SEQ IDNO: 41 and 42), R37P1C9 (SEQ ID NO: 43 and 44), R37P1H1 (SEQ ID NO: 45and 46), R42P3A9 (SEQ ID NO: 47 and 48), R43P3F2 (SEQ ID NO: 49 and 50),R43P3G5 (SEQ ID NO: 51 and 52), R59P2E7 (SEQ ID NO: 53 and 54), R11P3D3(SEQ ID NO: 55 and 56), R16P1C10 (SEQ ID NO: 57 and 58), R16P1E8 (SEQ IDNO: 59 and 60), R17P1A9 (SEQ ID NO: 61 and 62), R17P1D7 (SEQ ID NO: 63and 64), R17P1G3 (SEQ ID NO: 65 and 66), R17P2B6 (SEQ ID NO: 67 and 68),R11P3D3KE (SEQ ID NO: 69 and 70), R39P1C12 (SEQ ID NO: 71 and 72),R39P1F5 (SEQ ID NO: 73 and 74), R40P1C2 (SEQ ID NO: 75 and 76), R41P3E6(SEQ ID NO: 77 and 78), R43P3G4 (SEQ ID NO: 79 and 80), R44P3B3 (SEQ IDNO: 81 and 82), R44P3E7 (SEQ ID NO: 83 and 84), R49P2B7 (SEQ ID NO: 85and 86), R55P1G7 (SEQ ID NO: 87 and 88), or R59P2A7 (SEQ ID NO: 89 and90).

Table 2 shows examples of the peptides to which TCRs bind when thepeptide is in a complex with an MHC molecule.

TABLE 2 TCR name Peptide (SEQ ID NO:) R20P1H7, R7P1D5, R10P2G12KVLEHVVRV (SEQ ID NO: 216) R10P1A7 KIQEILTQV (SEQ ID NO: 124)R4P1D10, R4P3F9, R4P3H3 FLLDGSANV (SEQ ID NO: 239)R36P3F9, R52P2G11, R53P2A9 ILQDGQFLV (SEQ ID NO: 194)R26P1A9, R26P2A6, R26P3H1, KVLEYVIKV R35P3A4, R37P1C9, R37P1H1,(SEQ ID NO: 203) R42P3A9, R43P3F2, R43P3G5, R59P2E7R11KEA, R11P3D3, R16P1C10, SLLQHLIGL R16P1E8, R17P1A9, R17P1D7,(SEQ ID NO: 148) R17P1G3, R17P2B6, R11P3D3KE R39P1C12, R39P1F5, R40P1C2,ALSVLRLAL R41P3E6, R43P3G4, R44P3B3, (SEQ ID NO: 249)R44P3E7, R49P2B7, R55P1G7, R59P2A7

In an aspect, tumor associated antigen (TAA) peptides that are capableof use with the methods and embodiments described herein include, forexample, those listed in Table 4 and those TAA peptides described inU.S. Publication 20160187351, U.S. Publication 20170165335, U.S.Publication 20170035807, U.S. Publication 20160280759, U.S. Publication20160287687, U.S. Publication 20160346371, U.S. Publication 20160368965,U.S. Publication 20170022251, U.S. Publication 20170002055, U.S.Publication 20170029486, U.S. Publication 20170037089, U.S. Publication20170136108, U.S. Publication 20170101473, U.S. Publication 20170096461,U.S. Publication 20170165337, U.S. Publication 20170189505, U.S.Publication 20170173132, U.S. Publication 20170296640, U.S. Publication20170253633, U.S. Publication 20170260249, U.S. Publication 20180051080,and U.S. Publication No. 20180164315, the contents of each of thesepublications and sequence listings described therein are hereinincorporated by reference in their entireties.

In another aspect, the dimer Z1Z2 and/or the dimer Y1Y2 may be T celldimeric signaling modules, such as CD3δ/ε, CD3γ/ε, and CD247 ζ/ζ or ζ/η,a dimer of a TCRα variable region (Vα) and a TCRβ variable region (Vβ),a dimer of immunoglobulin heavy chain variable region (VH) andimmunoglobulin light chain variable region (VL), a dimer of Vα and VH, adimer of Vα and VL, a dimer of Vβ and VH, or a dimer of Vβ and VL.

In another aspect, the dimer Z1Z2 and/or the dimer Y1Y2 may be a TCRcoreceptor, such as a CD8a chain and CD8β chain, a CD4a chain and a CD4βchain, or any other suitable dimeric membrane receptors, preferablythose expressed in the CD8+ T cells and/or in the CD4+ T cells.

In some aspects, the dimer Z1Z2 is a TCR and the dimer Y1Y2 is a TCRcoreceptor.

Furin is a ubiquitous subtilisin-like proprotein convertase, whosenatural substrates include certain serum proteins and growth factorreceptors, such as the insulin-like growth factor receptor. Theconsensus sequence for furin cleavage is RXXR (SEQ ID NO: 93) but thepotential for actual cleavage is dependent on substrate tertiarystructure and the amino acids immediately surrounding the recognitionsite. Addition of a furin cleavage site plus the linker sequences (suchas GSG or SGSG (SEQ ID NO: 5)) may enable highly efficient geneexpression.

In one aspect, a nucleotide sequence of furin-linker-2A peptide arrangedin tandem may be positioned between Z1 and Z2, between Z1 and Y1,between Z1 and Y2, between Z2 and Y1, between Z2 and Y2, and/or betweenY1 and Y2. The furin may have a consensus sequence of RXXR (SEQ ID NO:93), e.g., RAKR (SEQ ID NO: 10). The linker sequence may be from 3 to 10amino acids long, such as from 3 to 8 amino acids long, from 3 to 5amino acids long, or from 3 to 4 amino acids long. In some aspects, thelinker sequence may be SGS, GGGS (SEQ ID NO: 257), GGGGS (SEQ ID NO:258), GGSGG (SEQ ID NO: 259), TVAAP (SEQ ID NO: 260), TVLRT (SEQ ID NO:261), TVSSAS (SEQ ID NO: 262). In some aspects, the linker sequence maybe GSG or SGSG (SEQ ID NO: 5). The 2A peptide may be selected from P2A(SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), F2A (SEQ ID NO:6), or any combination thereof.

In another aspect, a nucleotide sequence of linker-2A peptide arrangedin tandem may be positioned between Z1 and Z2, between Z1 and Y1,between Z1 and Y2, between Z2 and Y1, between Z2 and Y2, and/or betweenY1 and Y2. The linker sequence may be GSG or SGSG (SEQ ID NO: 5). The 2Apeptide may be selected from P2A (SEQ ID NO: 6), T2A (SEQ ID NO: 7), E2A(SEQ ID NO: 8), F2A (SEQ ID NO: 9), or any combination thereof.

Therapeutic Compositions

The invention also provides a therapeutic composition comprising apopulation of transduced cells, such as transduced T cells, describedherein.

The composition of the present disclosure may also include one or moreadjuvants. Adjuvants are substances that non-specifically enhance orpotentiate the immune response (e.g., immune responses mediated byCD8-positive T cells and helper-T (TH) cells to an antigen and wouldthus be considered useful in the medicament of the present invention.Suitable adjuvants include, but are not limited to, 1018 ISS, aluminumsalts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellinor TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31,Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2,IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivativesthereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2,MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vectorsystem, poly(lactide co-glycolide) [PLG]-based and dextranmicroparticles, talactoferrin SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21stimulon, which is derived from saponin, mycobacterial extracts andsynthetic bacterial cell wall mimics, and other proprietary adjuvantssuch as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's orGM-CSF are preferred. Several immunological adjuvants (e.g., MF59)specific for dendritic cells and their preparation have been describedpreviously (Allison and Krummel, 1995). Also cytokines may be used.Several cytokines have been directly linked to influencing dendriticcell migration to lymphoid tissues (e.g., TNF-), accelerating thematuration of dendritic cells into efficient antigen-presenting cellsfor T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, immune checkpoint inhibitors including ipilimumab, nivolumab,pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab,Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil,sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib,VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting keystructures of the immune system (e.g. anti-CD40, anti-TGFbeta,anti-TNFalpha receptor) and SC58175, which may act therapeuticallyand/or as an adjuvant. The amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, atezolizumab,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, and particulateformulations with poly(lactide co-glycolide) (PLG), virosomes, and/orinterleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, andIL-23.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

Methods of Manufacturing Polypeptides

The present invention also relates to a method of producing arecombinant host cell expressing a protein, such as a therapeuticprotein, said method comprising the steps consisting of: (i) introducingin vitro or ex vivo a vector of the invention into a competent hostcell, (ii) culturing in vitro or ex vivo the recombinant host cellobtained and (iii), optionally, selecting the cells which express and/orsecrete said protein.

The present invention also provides methods of manufacturingpolypeptides using vectors, such as the lentiviral transduction vectorsdisclosed herein, and the products of such methods. The methods cancomprise one or more of the following steps, e.g., transducing a hostcell with a lentivirus transduction vector to form a transduced hostcell, wherein said vector comprises an expressible heterologouspolynucleotide coding for a heterologous polypeptide of interest;culturing said transduced host cell under conditions effective toproduce said polypeptide of interest; isolating polypeptide from saidhost, e.g., from the culture medium when a polypeptide is secreted intothe culture medium. The heterologous polynucleotide sequence coding forthe polypeptide can comprise any further sequences necessary fortranscription, translation, and/or secretion into the medium (e.g.,secretory sequences). Any cells lines can be transduced in accordancewith the present invention, including, for example, CHO (such as CHODG44) and HEK 293 (such as HEK293F).

Transduction vectors can be prepared routinely, including according tothe methods described herein. For example, a producer cell line can betransformed with a helper plasmid (containing a suitable envelope andgag/pol precursor) and a transfer vector containing the heterologous NOIunder conditions effective to produce functional transduction vectors.The envelope protein can be selected for its ability to transduce atarget host cell in which the polypeptide is to be manufactured.

Examples of host cells, include, e.g., mammalian cell lines (e.g., Verocells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary orestablished cell cultures (e.g., produced from lymphoblasts,fibroblasts, embryonic cells, epithelial cells, nervous cells,adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cell (ATCCCRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which adihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) isdefective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.20 cell (ATCCCRL1662, hereinafter referred to as “YB2/0 cell”), and the like. TheYB2/0 cell may be preferred for some therapeutic antibodies, since ADCCactivity of chimeric or humanized antibodies is enhanced when expressedin this cell.

In an aspect, host cells may include T cells, such as CD4+ T cells, CD8+T cells, γδ T cells, and/or natural killer T cells.

In another aspect, host cells may include natural killer (NK) cells,dendritic cells, macrophages, and/or cancer cells.

In another aspect, host cells may not include NK cells.

In another aspect, host cells may not include cancer cells.

In particular, for expression of a therapeutic protein, such as adimeric therapeutic protein, the expression vector may be either of atype in which a gene encoding one polypeptide (chain) and a geneencoding the other polypeptide (chain) exists on separate vectors or ofa type in which both genes exist on the same vector (tandem type). Inrespect of easiness of construction of antibodies or TCR expressionvector, easiness of introduction into animal cells, and balance betweenthe expression levels of antibody H and L chains or alpha and betachains in animal cells, expression vector of the tandem type ispreferred (Shitara K et al. J Immunol Methods. 1994 Jan. 3;167(1-2):271-8).

For manufacturing flu vaccines, the following cell lines andcorresponding envelope proteins are preferred, e.g., HEK293 or CHO;VSV-G, ampho, Mokola, and Paramyxoviridae (for example, seencbi.nlm.nih.gov/ICTVdb/Ictv/fs_param.htm).

Any suitable or desired heterologous sequence can be expressed,including, e.g., vaccines, interferons (alpha, beta, gamma, epsilon),erythropoietin, Factor VIII, clotting factors, antibodies and fragmentsthereof (e.g., including single chain, Fab, and humanized), insulin,chemokines, cytokines, growth factors, angiogenesis modulatory factors,apoptosis modulatory factors, etc. Single-chain antibodies (e.g., singlechain variable fragments or “scFv”) can be made routinely.

In certain embodiments of the present invention, lentiviral transductionvectors can be used to prepare antigenic preparations that be used asvaccines. Any suitable antigen(s) can be prepared in accordance with thepresent invention, including antigens obtained from prions, viruses,mycobacterium, protozoa (e.g., Plasmodium falciparum (malaria)),trypanosomes, bacteria (e.g., Streptococcus, Neisseria, etc.), etc.

Host cells can be transduced with a single lentiviral vector containingone or more heterologous NOI(s), or with a plurality of lentiviralvectors, where each vector comprises the same or different heterologousNOI(s). For example, a multi-subunit antigen (including intracellularand cell-surface multi-subunit components) can be prepared by expressingthe individual subunits on separate vectors, but infecting the same hostcell with all the vectors, such that assembly occurs within the hostcell.

Vaccines often contain a plurality of antigen components, e.g., derivedfrom different proteins, and/or from different epitopic regions of thesame protein. For example, a vaccine against a viral disease cancomprise one or more polypeptide sequences obtained from the viruswhich, when administered to a host, elicit an immunogenic or protectiveresponse to viral challenge.

As mentioned, the present invention can also be used to preparepolypeptide multimers, e.g., where an antigenic preparation is producedwhich is comprised of more than one polypeptide. For instance, viruscapsids can be made up of more than one polypeptide subunit. Bytransducing a host cell with vectors carrying different viral envelopesequences, the proteins, when expressed in the cell, can self-assembleinto three-dimensional structures containing more than one proteinsubunit (e.g., in their native configuration). The structures canpossess functional activity, including antigenic activity, enzymeactivity, cell binding activity, etc. Moreover, when expressed in asuitable cell line, they can be secreted into the cell culture medium,facilitating purification. For instance, when influenza N and H capsidproteins, and optionally M protein (see below), are introduced into aproduction cell line using lentiviral transduction vectors, emptycapsids or viral-like particles (VLP) can be formed in the cell, andthen secreted into the culture media. Such VLP can be routinely isolatedand purified, and then administered as an influenza vaccine. A VLP is,e.g., a self-assembled capsid which does not contain substantial amounts(e.g., is empty) of viral RNA. A VLP is preferably able to elicit animmune response that is effective to provide at least some degree ofprotection against a challenge of the native infectious virus particle,or at least elicit antibodies to it.

Currently, there are many available viral vaccines, including vaccinesto such diseases as measles, mumps, hepatitis (A and B), rubella,influenza, polio, smallpox, varicella, adenovirus, Japaneseencephalitis, rabies, ebola, etc. The present invention can be used toprepare vaccines against any of the above-mentioned diseases.

Examples of viruses to which vaccines can be produced in accordance withthe present invention include, e.g., orthomyxoviruses, influenza virus A(including all strains varying in their HA and NA proteins, such as(non-limiting examples) H1N1, H1N2, H2N2, H3N2, H7N7, and H3N8);influenza B, influenza C, thogoto virus (including Dhori, Batken virus,SiAR 126 virus), and isavirus (e.g., infectious salmon anemia virus).These include influenza isolated or transmitted from all species types,including isolates from invertebrates, vertebrates, mammals, humans,non-human primates, monkeys, pigs, cows, and other livestock, birds,domestic poultry such as turkeys, chickens, quail, and ducks, wild birds(including aquatic and terrestrial birds), reptiles, etc. These alsoinclude existing strains which have changed, e.g., through mutation,antigenic drift, antigenic shift, recombination, etc., especiallystrains which have increased virulence and/or interspecies transmission(e.g., human-to-human).

Of particular interest are influenza viruses which are panzootic and/orwhich cross species either because they have a broad host range, orbecause of recombination in the infected host, and/or because ofnaturally-occurring or directed mutation. For example, H5N1 (inreference to the subtypes of surface antigens present on the virus,hemagglutinin type 5 and neuraminadase type 1) is a subtype of avianinfluenza A, which caused an outbreak of flu in domestic birds in Asia.As of November 2005, more 120 million birds died from infection or werekilled to prevent further infection from spreading. This virus has alsospread into human hosts (“bird flu”) where it is associated with highlethality.

An influenza antigenic preparation (such as a vaccine) can comprise oneor more polypeptides that occur naturally in an influenza virion.However, it preferably does not comprise all the polypeptide genes thatwould give rise to the native pathogenic virus. These include, e.g.,hemagglutinin (encoded by HA gene), neuraminidase (encoded by NA gene),nucleoprotein (encoded by NA gene), matrix (M1) proteins (encoded by Mgene), M2 (encoded by M gene), non-structural proteins (encoded by NSgene), and polymerases. The naturally-occurring virion is sheathed in alipid bilayer which is “studded” with integral proteins H and N (“capsidlayer”). Matrix proteins (M1) form a protein layer (“matrix layer”)underneath the viral membrane, and are involved in viral assembly,stability and integrity. See, e.g., Harris et al., Virol. 289:34-44,2001. M2 protein is a membrane protein ion channel. A VLP of the presentinvention can comprise H, N, and optionally M1 and M2 proteins.Sequences for said proteins are known in the art and/or can beidentified in GenBank. See, e.g., Widjaja et al. J. Virol.,78:8771-8779, 2004 for M1 and M2 sequences.

These can be cloned into transfer vectors, either individually or on thesame plasmid, and utilized to produce transduction vectors. In oneembodiment of the present invention, a plurality of transduction vectorscan be prepared, each which contains a unique influenza gene sequence(e.g., coding for H, for N, and for M1 to result in a three differenttransduction vectors). When such vectors are co-expressed in the samehost cell (e.g., CHO or HEK293), a self-assembling VLP is produced whichcan be secreted into the medium, harvested by centrifugation, and thenadministered as a vaccine.

Transduction vectors of the present invention can result in high levelsof heterologous protein production, e.g., from about 0.1 to 0.3 mg/ml toabout 5-10 mg/ml, or more, of recombinant heterologous protein per ml ofunprocessed culture media, when such proteins are secreted into theculture media.

The present application also provides methods of producing antibodies.For example, methods are provided to produce monoclonal antibodies(e.g., human, mouse, and other mammalian types) without the need forhybridomas or animal models. In one non-limiting example, lentiviralvectors expressing oncogenic proteins are transduced in peripheral bloodB cells from mice previously stimulated with antigen. These vectorsefficiently transduce the mouse cells to make them into antibodyproducing cells. In a second non-limiting example, two lentiviralvectors are engineered, one expressing the heavy antibody chain and thesecond vector the light antibody chain. The constant areas of the genesare derived from the human (or other species if desired) immunoglobulingene (e.g., IgG, IgM or other type of Ig). The variable areas of thegenes are modified or degenerated to create diversity. The degeneratesequence can be obtained by any suitable techniques that is known in theart and cloned into the lentiviral vector to create a library oflentiviral vectors that express either the heavy or light immunoglobulinmolecules. The antibodies can be produced by transducing cells with bothvectors to produce functional antibodies that contain both heavy andlight chains. Transduced and expressing cells can be selected andscreened for binding to antigen, and then positive clones can beisolated and subjected to multiple rounds of affinity maturation.

An advantage of this method is that antibodies are produced in anon-biased method. Other methods, such as traditional hybridoma andXenomouse technologies rely on B cells that have undergone clonalselection and deletion of particular antibody clones since they arereactive to endogenous, for example, mouse tissue. Some of these deletedclones may be valuable as antibodies as they could cross react withhuman antigens. The advantage of the described method is that there isno deletion of molecular antibody clones and they are all analyzed in anon-biased method and yet are fully humanized (if humanization isdesired) antibody molecules. Another advantage of lentiviral vectors isthat the genes can be transduced into cells at high multiplicity toproduce a variety of antibody type in one cell. This reduces the numberof cells that need to be produced to create a library that contains avery diverse antigenic binding sites. A second advantage is placing theheavy and light genes in different lentiviral vectors so that additionaldiversity can be generated by transducing cells with a highermultiplicity of infection than 1. For example, if a MOI of 10 is usedfor the transduction of cells with each heavy and light chain expressingLentiviral vector, then the number of combinations of antibodiesproduced in each cell is 100. Therefore in a 96-well plate, where thereare about 10,000 cells in a single well, the number of possible variantsthat can be generated with this method is 1,000,000 in a single well ofa 96-well plate. Therefore, with scale, a large number of antibodyvariants can be generated with this method. The method does not limit tousing a MOI of 10 for each construct per cell, higher MOIs can also beused, as needed. For example, if a MOI of 100 is used then each cell canproduce 10,000 variant antibodies and each well of a 96 well plate canproduce 10,000,000,000 variants. Therefore, each 96 well plate canproduce 1×10¹² variant antibody molecules that can be used for screeningagainst a target antigen, for which there are many methods known in theart (e.g., ELISA). Once a particular well has been identified thatproduces the desired antibody reaction, then the cells can be cloned bylimiting dilution to find the cell clone that expresses the correctantibody. Once this clone has been identified, then PCR can be used toclone out the vectors that express the heavy and light antibody chains.The vector DNA can then be transfected with helper construct(s) toproduce vector. Alternatively, this clone of cells can be transfecteddirectly with the helper construct(s) (PEI, calcium phosphate,lipotransfection, or other transfection method known in the art), toproduce the variant lentiviral vectors. The vectors that are producedcan then tittered and then transduced onto cells at a lower MOI, but alarger number of cells, to isolate a clone that produces the antibody ofinterest. Once the clone of cell is isolated, then the antibody can beproduced to higher titers by transducing cells with higher multiplicityof infection, the same method is not limited to whole antibody moleculesbut can also be applied to single chain antibodies, antibody fragments,phage display and other antibody-like molecules, all known in the art.In addition to expressing the antibody, the vector can express othergenes to increase the production of the monoclonal antibody, or toincrease their yield. Such genes can be oncogenes such as ras and myc,but other genes can also be used, such as anti-apoptotic genes such asBcl-2. Furthermore, such vectors can be used to create monoclonalantibodies from B cells in the blood of animals that have been exposedto antigen. For example, B cells from mice exposed to antigen can betransformed into myeloma cells by using a combination of oncogenes orgene silencing RNA. Such genes include, e.g., Growth Factors, including,e.g., Amphiregulin, B-lymphocyte stimulator, Interleukin 16 (IL16),Thymopoietin, TRAIL, Apo-2, Pre B cell colony enhancing factor,Endothelial differentiation-related factor 1 (EDF1), Endothelialmonocyte activating polypeptide II, Macrophage migration inhibitoryfactor MIF, Natural killer cell enhancing factor (NKEFA), Bonemorphogenetic protein 8 (osteogenic protein 2), Bone morphogenic protein6, Connective tissue growth factor (CTGF), CGI-149 protein(neuroendocrine differentiation factor), Cytokine A3 (macrophageinflammatory protein 1-alpha), Glialblastoma celldifferentiation-related protein (GBDR1), Hepatoma-derived growth factor,Neuromedin U-25 precursor, any tumor gene, oncogene, proto-oncogene orcell modulating gene (which can be found atcondor.bcm.tmc.edu/oncogene), Vascular endothelial growth factor (VEGF),Vascular endothelial growth factor B (VEGF-B), T-cell specific RANTESprecursor, Thymic dendritic cell-derived factor 1; Receptors, such asActivin A receptor, type II (ACVR2), β-signal sequence receptor (SSR2),CD14 monocyte LPS receptor, CD36 (collagen type 1/thrombospondinreceptor)-like 2, CD44R (Hermes antigen gp90 homing receptor), G proteincoupled receptor 9, Chemokine C×C receptor 4, Colony stimulating factor2 receptor β(CSF2RB), FLT-3 receptor tyrosine kinase, Similar totransient receptor potential C precursor, Killer cell lectin-likereceptor subfamily B, Low density lipoprotein receptor gene,low-affinity Fc-gamma receptor IIC, MCP-1 receptor, Monocytechemoattractant protein 1 receptor (CCR2), Nuclear receptor subfamily 4,group A, member 1, Orphan G protein-coupled receptor GPRC5D, Peroxisomeproliferative activated receptor gamma, Pheromore related-receptor(rat), Vasopressin-activated calcium mobilizing putative receptor,Retinoic x receptor, Toll-like receptor 6, Transmembrane activator andCAML interactor (TACI), B cell maturation peptide (BCMA), CSF-1receptor, Interferon (α, β and gamma) receptor 1 (IFNAR1).

Methods of Treatment

The vectors provided herein can be used in a wide variety of therapeuticmethods.

In some aspects, a lentiviral vector for therapeutic use is providedwhich expresses a native or fusion polypeptide comprising of anyindividual or combination of a human chemokine and a viral or bacterialantigen (e.g. HIV, diphtheria toxin antigen), a chemokine (e.g. IP-10,MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC) or apro-apoptotic protein, a suicide gene protein or a protein that promotesthe inflammatory response.

In addition, the present invention provides a method of producing animmune response in a subject, comprising administering to the subjectany of the individual or fusion polypeptides of this invention, such asa chemokine and a human immunodeficiency virus (HIV) antigen, or achemokine, a pro-apoptotic gene, a suicide gene and a tumor antigen,either as a protein or a nucleic acid encoding the individual or fusionpolypeptide expressed from a lentiviral vector. Also provided is amethod of treating a cancer in a subject comprising administering to thesubject a lentiviral vector expressing any of the individual or fusionpolypeptides of this invention, such as a chemokine and a tumor antigen,either as a protein or a nucleic acid encoding the fusion polypeptide.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject any combination ofthe following peptides derived from the following proteins: chemokine,suicide gene, HIV protein, cytokine, cell surface protein, tumorantigen, or any cellular gene that affects the production of HIV fromthe cell (either by overexpressing the cellular gene or inhibiting itsexpression by RNAi, or the like), all provided and expressed from alentiviral vector.

In some aspects, compositions containing engineered immune cells, suchas T cells (e.g., γδ T cells), described herein may be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,pharmaceutical compositions can be administered to a subject alreadysuffering from a disease or condition in an amount sufficient to cure orat least partially arrest the symptoms of the disease or condition. Anengineered immune cell can also be administered to lessen a likelihoodof developing, contracting, or worsening a condition. Effective amountsof a population of engineered immune cells for therapeutic use can varybased on the severity and course of the disease or condition, previoustherapy, the subject's health status, weight, and/or response to thedrugs, and/or the judgment of the treating physician.

Engineered immune cells, such as T cells, of the present disclosure canbe used to treat a subject in need of treatment for a condition, forexample, a cancer described herein.

A method of treating a condition (e.g., ailment) in a subject withengineered immune cells, such as engineered T cells, may includeadministering to the subject a therapeutically effective amount ofengineered immune cells, such as engineered T cells. Engineered immunecells, such as engineered T cells, of the present disclosure may beadministered at various regimens (e.g., timing, concentration, dosage,spacing between treatment, and/or formulation). A subject can also bepreconditioned with, for example, chemotherapy, radiation, or acombination of both, prior to receiving engineered immune cells, such asengineered T cells, of the present disclosure. A population ofengineered immune cells, such as engineered T cells, may also be frozenor cryopreserved prior to being administered to a subject. A populationof engineered immune cells, such as engineered T cells, can include twoor more cells that express identical, different, or a combination ofidentical and different tumor recognition moieties. For instance, apopulation of engineered immune cells, such as engineered T-cells, caninclude several distinct engineered immune cells, such as engineered Tcells, that are designed to recognize different antigens, or differentepitopes of the same antigen.

Engineered immune cells, such as engineered T cells, of the presentdisclosure may be used to treat various conditions. In an aspect,engineered immune cells, such as engineered T cells, of the presentdisclosure may be used to treat a cancer, including solid tumors andhematologic malignancies. Non-limiting examples of cancers include:acute lymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancers, brain tumors, such ascerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoma of unknown primary origin, centralnervous system lymphoma, cerebellar astrocytoma, cervical cancer,childhood cancers, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, desmoplastic small round cell tumor, endometrialcancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ celltumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia,head and neck cancer, heart cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, isletcell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip andoral cavity cancer, liposarcoma, liver cancer, lung cancers, such asnon-small cell and small cell lung cancer, lymphomas, leukemias,macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanomas, mesothelioma, metastatic squamous neckcancer with occult primary, mouth cancer, multiple endocrine neoplasiasyndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer, ovarian germ celltumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineal astrocytoma, pineal germinoma,pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell carcinoma, renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skincancers, skin carcinoma merkel cell, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma,throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastictumor (gestational), cancers of unknown primary site, urethral cancer,uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrmmacroglobulinemia, and Wilms tumor.

In an aspect, engineered immune cells, such as engineered T cells, ofthe present disclosure may be used to treat an infectious disease. Inanother aspect, engineered immune cells, such as engineered T cells, ofthe present disclosure may be used to treat an infectious disease, aninfectious disease may be caused a virus. In yet another aspect,engineered immune cells, such as engineered T cells, of the presentdisclosure may be used to treat an immune disease, such as an autoimmunedisease.

Treatment with engineered immune cells, such as engineered T cells, ofthe present disclosure may be provided to the subject before, during,and after the clinical onset of the condition. Treatment may be providedto the subject after 1 day, 1 week, 6 months, 12 months, or 2 yearsafter clinical onset of the disease. Treatment may be provided to thesubject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10years or more after clinical onset of disease. Treatment may be providedto the subject for less than 1 day, 1 week, 1 month, 6 months, 12months, or 2 years after clinical onset of the disease. Treatment mayalso include treating a human in a clinical trial. A treatment caninclude administering to a subject a pharmaceutical compositioncomprising engineered immune cells, such as engineered T cells, of thepresent disclosure.

In another aspect, administration of engineered immune cells, such asengineered T cells, of the present disclosure to a subject may modulatethe activity of endogenous lymphocytes in a subject's body. In anotheraspect, administration of engineered immune cells, such as engineered Tcells, to a subject may provide an antigen to an endogenous T-cell andmay boost an immune response. In another aspect, the memory T cell maybe a CD4+ T-cell. In another aspect, the memory T cell may be a CD8+T-cell. In another aspect, administration of engineered immune cells,such as engineered T cells, of the present disclosure to a subject mayactivate the cytotoxicity of another immune cell. In another aspect, theother immune cell may be a CD8+ T-cell. In another aspect, the otherimmune cell may be a Natural Killer T-cell. In another aspect,administration of engineered immune cells, such as engineered T-cells,of the present disclosure to a subject may suppress a regulatory T-cell.In another aspect, the regulatory T-cell may be a FOX3+ Treg cell. Inanother aspect, the regulatory T-cell may be a FOX3-Treg cell.Non-limiting examples of cells whose activity can be modulated byengineered immune cells, such as engineered T cells of the disclosuremay include: hematopioietic stem cells; B cells; CD4; CD8; red bloodcells; white blood cells; dendritic cells, including dendritic antigenpresenting cells; leukocytes; macrophages; memory B cells; memoryT-cells; monocytes; natural killer cells; neutrophil granulocytes;T-helper cells; and T-killer cells.

During most bone marrow transplants, a combination of cyclophosphamidewith total body irradiation may be conventionally employed to preventrejection of the hematopietic stem cells (HSC) in the transplant by thesubject's immune system. In an aspect, incubation of donor bone marrowwith interleukin-2 (IL-2) ex vivo may be performed to enhance thegeneration of killer lymphocytes in the donor marrow. Interleukin-2(IL-2) is a cytokine that may be necessary for the growth,proliferation, and differentiation of wild-type lymphocytes. Currentstudies of the adoptive transfer of γδ T-cells into humans may requirethe co-administration of γδ T-cells and interleukin-2. However, bothlow- and high-dosages of IL-2 can have highly toxic side effects. IL-2toxicity can manifest in multiple organs/systems, most significantly theheart, lungs, kidneys, and central nervous system. In another aspect,the disclosure provides a method for administrating engineered γδ Tcells to a subject without the co-administration of a native cytokine ormodified versions thereof, such as IL-2, IL-15, IL-12, IL-21. In anotheraspect, engineered γδ T cells can be administered to a subject withoutco-administration with IL-2. In another aspect, engineered γδ T cellsmay be administered to a subject during a procedure, such as a bonemarrow transplant without the co-administration of IL-2.

Methods of Administration

One or multiple engineered immune cell, such as engineered T cell,populations may be administered to a subject in any order orsimultaneously. If simultaneously, the multiple engineered immune cell,such as T cell, can be provided in a single, unified form, such as anintravenous injection, or in multiple forms, for example, as multipleintravenous infusions, s.c, injections or pills. Engineered immunecells, such as engineered T-cells, can be packed together or separately,in a single package or in a plurality of packages. One or all of theengineered immune cells, such as engineered T cells, can be given inmultiple doses. If not simultaneous, the timing between the multipledoses may vary to as much as about a week, a month, two months, threemonths, four months, five months, six months, or about a year. Inanother aspect, engineered immune cells, such as engineered T cells, canexpand within a subject's body, in vivo, after administration to asubject. Engineered immune cells, such as engineered T cells, can befrozen to provide cells for multiple treatments with the same cellpreparation. Engineered immune cells, such as engineered T cells, of thepresent disclosure, and pharmaceutical compositions comprising the same,can be packaged as a kit. A kit may include instructions (e.g., writteninstructions) on the use of engineered immune cells, such as engineeredT cells, and compositions comprising the same.

In another aspect, a method of treating a cancer comprises administeringto a subject a therapeutically-effective amount of engineered immunecells, such as engineered T cells, in which the administration treatsthe cancer. In another embodiments, the therapeutically-effective amountof engineered immune cells, such as engineered T cells, may beadministered for at least about 10 seconds, 30 seconds, 1 minute, 10minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months,6 months, or 1 year. In another aspect, the therapeutically-effectiveamount of the engineered immune cells, such as engineered T cells, maybe administered for at least one week. In another aspect, thetherapeutically-effective amount of engineered immune cells, such asengineered T cells, may be administered for at least two weeks.

Engineered immune cells, such as engineered T-cells, described hereincan be administered before, during, or after the occurrence of a diseaseor condition, and the timing of administering a pharmaceuticalcomposition containing an engineered immune cells, such as engineeredT-cell, can vary. For example, engineered immune cells, such asengineered T cells, can be used as a prophylactic and can beadministered continuously to subjects with a propensity to conditions ordiseases in order to lessen the likelihood of occurrence of the diseaseor condition. Engineered immune cells, such as engineered T-cells, canbe administered to a subject during or as soon as possible after theonset of the symptoms. The administration of engineered immune cells,such as engineered T cells, can be initiated immediately within theonset of symptoms, within the first 3 hours of the onset of thesymptoms, within the first 6 hours of the onset of the symptoms, withinthe first 24 hours of the onset of the symptoms, within 48 hours of theonset of the symptoms, or within any period of time from the onset ofsymptoms. The initial administration can be via any route practical,such as by any route described herein using any formulation describedherein. In another aspect, the administration of engineered immunecells, such as engineered T cells, of the present disclosure may be anintravenous administration. One or multiple dosages of engineered immunecells, such as engineered T cells, can be administered as soon as ispracticable after the onset of a cancer, an infectious disease, animmune disease, sepsis, or with a bone marrow transplant, and for alength of time necessary for the treatment of the immune disease, suchas, for example, from about 24 hours to about 48 hours, from about 48hours to about 1 week, from about 1 week to about 2 weeks, from about 2weeks to about 1 month, from about 1 month to about 3 months. For thetreatment of cancer, one or multiple dosages of engineered immune cells,such as engineered T cells, can be administered years after onset of thecancer and before or after other treatments. In another aspect,engineered immune cells, such as engineered T cells, can be administeredfor at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least3 months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 12 months, at least 1 year, atleast 2 years at least 3 years, at least 4 years, or at least 5 years.The length of treatment can vary for each subject.

Preservation

In an aspect, immune cells, such as T cells, may be formulated infreezing media and placed in cryogenic storage units such as liquidnitrogen freezers (−196° C.) or ultra-low temperature freezers (−65° C.,−80° C., −120° C., or −150° C.) for long-term storage of at least about1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2years, 3 years, or at least 5 years. The freeze media can containdimethyl sulfoxide (DMSO), and/or sodium chloride (NaCl), and/ordextrose, and/or dextran sulfate and/or hydroyethyl starch (HES) withphysiological pH buffering agents to maintain pH between about 6.0 toabout 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 toabout 8.0 or about 6.5 to about 7.5. The cryopreserved immune cells,such as T cells, can be thawed and further processed by stimulation withantibodies, proteins, peptides, and/or cytokines as described herein.The cryopreserved immune cells, such as T-cells, can be thawed andgenetically modified with viral vectors (including retroviral,adeno-associated virus (AAV), and lentiviral vectors) or non-viral means(including RNA, DNA, e.g., transposons, and proteins) as describedherein. The modified immune cells, such as modified T cells, can befurther cryopreserved to generate cell banks in quantities of at leastabout 1, 5, 10, 100, 150, 200, 500 vials at about at least 10¹, 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or at least about 10¹⁰ cells per mLin freeze media. The cryopreserved cell banks may retain theirfunctionality and can be thawed and further stimulated and expanded. Inanother aspect, thawed cells can be stimulated and expanded in suitableclosed vessels, such as cell culture bags and/or bioreactors, togenerate quantities of cells as allogeneic cell product. Cryopreservedimmune cells, such as T cells, can maintain their biological functionsfor at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 13 months, 15 months, 18 months, 20 months, 24months, 30 months, 36 months, 40 months, 50 months, or at least about 60months under cryogenic storage condition. In another aspect, nopreservatives may be used in the formulation. Cryopreserved immunecells, such as T-cells, can be thawed and infused into multiple patientsas allogeneic off-the-shelf cell product.

In an aspect, engineered immune cells, such as engineered T-cells,described herein may be present in a composition in an amount of atleast 1×10³ cells/ml, at least 2×10³ cells/ml, at least 3×10³ cells/ml,at least 4×10³ cells/ml, at least 5×10³ cells/ml, at least 6×10³cells/ml, at least 7×10³ cells/ml, at least 8×10³ cells/ml, at least9×10³ cells/ml, at least 1×10⁴ cells/ml, at least 2×10⁴ cells/ml, atleast 3×10⁴ cells/ml, at least 4×10⁴ cells/ml, at least 5×10⁴ cells/ml,at least 6×10⁴ cells/ml, at least 7×10⁴ cells/ml, at least 8×10⁴cells/ml, at least 9×10⁴ cells/ml, at least 1×10⁵ cells/ml, at least2×10⁵ cells/ml, at least 3×10⁵ cells/ml, at least 4×10⁵ cells/ml, atleast 5×10⁵ cells/ml, at least 6×10⁵ cells/ml, at least 7×10⁵ cells/ml,at least 8×10⁵ cells/ml, at least 9×10⁵ cells/ml, at least 1×10⁶cells/ml, at least 2×10⁶ cells/ml, at least 3×10⁶ cells/ml, at least4×10⁶ cells/ml, at least 5×10⁶ cells/ml, at least 6×10⁶ cells/ml, atleast 7×10⁶ cells/ml, at least 8×10⁶ cells/ml, at least 9×10⁶ cells/ml,at least 1×10⁷ cells/ml, at least 2×10⁷ cells/ml, at least 3×10⁷cells/ml, at least 4×10⁷ cells/ml, at least 5×10⁷ cells/ml, at least6×10⁷ cells/ml, at least 7×10⁷ cells/ml, at least 8×10⁷ cells/ml, atleast 9×10⁷ cells/ml, at least 1×10⁸ cells/ml, at least 2×10⁸ cells/ml,at least 3×10⁸ cells/ml, at least 4×10⁸ cells/ml, at least 5×10⁸cells/ml, at least 6×10⁸ cells/ml, at least 7×10⁸ cells/ml, at least8×10⁸ cells/ml, at least 9×10⁸ cells/ml, at least 1×10⁹ cells/ml, ormore, from about 1×10³ cells/ml to about at least 1×10⁸ cells/ml, fromabout 1×10⁵ cells/ml to about at least 1×10⁸ cells/ml, or from about1×10⁶ cells/ml to about at least 1×10⁸ cells/ml.

In an aspect, methods described herein may be used to produce autologousor allogenic products according to an aspect of the disclosure.

EXAMPLES Example 1

TABLE 3 DNA and protein sequences SEQ ID NO: Description Sequence 1WT WPRE gagcatcttaccgccatttatacccatatt derived fromtgttctgtttttcttgatttgggtatacat GenBank ttaaatgttaataaaacaaaatggtggggcAccession No. aatcatttacattttatgggatatgtaatt J02440.1actagttcaggtgtattgccacaagacaaa catgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggattac aaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtgga tatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcc tccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaa cgtggcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccacc acctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactc atcgccgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataattcc gtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccaactgg atcctgcgcgggacgtccttctgctacgtcccttcggctctcaatccagcggacctccct tcccgaggccttctgccggttctgcggcctctcccgcgtcttcgctttcggcctccgacg agtcggatctccctttgggccgcctccccg cctg 2WT WPRE cagtctgacgtacgcgtaatcaacctctgg derived fromattacaaaatttgtgaaagattgactggta GenBank ttcttaactatgttgctccttttacgctatAccession No. gtggatacgctgctttaatgcctttgtatc J04514.1atgctattgcttcccgtatggctttcattt tctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtca ggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattg ccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcgg aactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgaca attccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgcca cctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacc ttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctc agacgagtcggatctccctttgggccgcct ccccgcc 3mutant WPRE gagcatcttaccgccatttatacccatatttgttctgtttttcttgatttgggtatacat ttaaatgttaataaaacaaaatggtggggcaatcatttacattttttgggatatgtaatt actagttcaggtgtattgccacaagacaaacttgttaagaaactttcccgttatttacgc tctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattctt aactttgttgctccttttacgctgtgtggatttgctgctttattgcctctgtatcttgct attgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctctt tttgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgac gcaacccccactggctggggcattgccaccacctgtcaactcctttctgggactttcgct ttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggaca ggggctaggttgctgggcactgataattccgtggtgttgtc 4 mutant WPRE cagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggta ttcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatc atgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgt ctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttg ctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactt tcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgt cctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgct acgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgc ggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcct ccccgcc 5 Linker SGSG 6 P2AATNFSLLKQAGDVEENPGP 7 T2A EGRGSLLTCGDVEENPGP 8 E2A QCTNYALLKLAGDVESNPGP9 F2A VKQTLNFDLLKLAGDVESNPGP 10 Furin RAKR 11 CD8 alphaMALPVTALLLPLALLLHAARPSQFRVSPLD chain RTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFS GKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN RRRVCKCPRPVVKSGDKPSLSARYV 12 CD8 betaMRPRLWLLLAAQLTVLHGNSVLQQTPAYIK chain VQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKI AVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTL KKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQPQG EGISGTFVPQCLHGYYSNTTTSQKLLNPWI LKT 13R11KEA alpha MEKNPLAAPLLILWFHLDCVSSILNVEQSP chainQSLHVQEGDSTNFTCSFPSSNFYALHWYRK ETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDM RFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 14R11KE beta MDSWTFCCVSLCILVAKHTDAGVIQSPRHE chainVTEMGQEVTLRCKPISGHNSLFWYRETMMR GLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDT QYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 15 R20P1H7MEKMLECAFIVLWLQLGWLSGEDQVTQSPE alpha chainALRLQEGESSSLNCSYTVSGLRGLFWYRQD PGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQGENSGYST LTFGKGTMLLVSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS 16R20P1H7 beta MGPQLLGYVVLCLLGAGPLEAQVTQNPRYL chainITVTGKKLTVTCSQNMNHEYMSWYRQDPGL GLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLGPGLAA YNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLS ENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS ALVLMAMVKRKDSRG 17 R7P1D5 alphaMKTFAGFSFLFLWLQLDCMSRGEDVEQSLF chain LSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKD KHLSLRIADTQTGDSAIYFCAEYSSASKIIFGSGTRLSIRPNIQNPDPAVYQLRDSKSSD KSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNN SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW SS 18 R7P1D5 betaMGSWTLCCVSLCILVAKHTDAGVIQSPRHE chain VTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPN ASFSTLKIQPSEPRDSAVYFCASRANTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEA EISHTQKATLVCLATGFYPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSR LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQ QGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG19 R10P2G12 MLTASLLRAVIASICVVSSMAQKVTQAQTE alpha chainISVVEKEDVTLDCVYETRDTTYYLFWYKQP PSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEGNSG NTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLSVIGFRILLLKVAGFNLLM TLRLWSS 20R10P2G12 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYL beta chainVKRTGEKVFLECVQDMDHENMFWYRQDPGL GLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSSGSHQ ETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWW WNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDSRG 21 R10P1A7MKTFAGFSFLFLWLQLDCMSRGEDVEQSLF alpha chainLSVREGDSSVINCTYTDSSSTYLYWYKQEP GAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESKETRLMF GDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVL DMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNL NFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 22R10P1A7 beta MLLLLLLLGPGISLLLPGSLAGSGLGAWSQ chainHPSVWICKSGTSVKIECRSLDFQATTMFWY RQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSA RAGGHEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVE LSWVWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYG LSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDSRG 23 R4P1D10MKSLRVLLVILWLQLSWWWSQQKEVEQNSG alpha chainPLSVPEGAIASLNCTYSDRGSQSFFWYRQY SGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFHDKIIF GKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVL DMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNL NFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 24R4P1D10 beta MGFRLLCCVAFCLLGAGPVDSGVTQTPKHL chainITATGQRVTLRCSPRSGDLSVYWYQQSLDQ GLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVASAYGY TFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWWNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDF 25 R4P3F9 alphaMKSLRVLLVILWLQLSWWWSQQKEVEQNSG chain PLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKAS QYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKS SDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAF NNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS 26 R4P3F9 betaMGFRLLCCVAFCLLGAGPVDSGVTQTPKHL chain ITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPD LHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEA EISHTQKATLVCLATGFFPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSR LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQ QGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDF27 R4P3H3 alpha MKSLRVLLVILWLQLSWWWSQQKEVEQNSG chainPLSVPEGAIASLNCTYSDRGSQSFFWYRQY SGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKAGNQFYF GTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVL DMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNL NFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 28R4P3H3 beta MGTRLLCWWVLGFLGTDHTGAGVSQSPRYK chainVAKRGQDVALRCDPISGHVSLFWYQQALGQ GPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLLTSGG DNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG 29 R36P3F9METLLGVSLVILWLQLARVNSQQGEEDPQA alpha chainLSIQEGENATMNCSYKTSINNLQWYRQNSG RGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATVSNYQLIWG AGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLN FQNLSVIGFRILLLKVAGFNLLMTLRLWSS 30R36P3F9 beta MGPQLLGYVVLCLLGAGPLEAQVTQNPRYL chainITVTGKKLTVTCSQNMNHEYMSWYRQDPGL GLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSSTSGGLS GETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG 31 R52P2G11MKKHLTTFLVILWLYFYRGNGKNQVEQSPQ alpha chainSLIILEGKNCTLQCNYTVSPFSNLRWYKQD TGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSAYGKLQ FGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLSVIGFRILLLKVAGFNLLMTLRLW SS 32R52P2G11 MDSWTFCCVSLCILVAKHTDAGVIQSPRHE beta chainVTEMGQEVTLRCKPISGHNSLFWYRQTMMR GLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLGSPDG NQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSW WWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDF 33 R53P2A9MACPGFLWALVISTCLEFSMAQTVTQSQPE alpha chainMSVQEAETVTLSCTYDTSESDYYLFWYKQP PSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYNSYAGG TSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDV YITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEK SFETDTNLNFQNLSVIGFRILLLKVAGFNL LMTLRLWSS34 R53P2A9 beta MGPGLLCWWLLCLLGAGPVDAGVTQSPTHL chainIKTRGQQVTLRCSPISGHKSVSWYQQVLGQ GPQFIFQYYEKEERGRGNFPDRFSARQFPNYSSELNVNALLLGDSALYLCASSLDGTSEQ YFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 35 R26P1A9METLLGVSLVILWLQLARVNSQQGEEDPQA alpha chainLSIQEGENATMNCSYKTSINNLQWYRQNSG RGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCLIGASGSRLTF GEGTQLTVNPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVL DMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNL NFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 36R26P1A9 beta MGSWTLCCVSLCILVAKHTDAGVIQSPRHE chainVTEMGQEVTLRCKPISGHDYLFWYRQTMMR GLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSYFGWNE KLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWW NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDF 37 R26P2A6MMKSLRVLLVILWLQLSWVWSQQKEVEQDP alpha chainGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQ YSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDVSGGY NKLIFGAGTRLAVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS 38R26P2A6 beta MGPQLLGYVVLCLLGAGPLEAQVTQNPRYL chainITVTGKKLTVTCSQNMNHEYMSWYRQDPGL GLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASTTPDGTDE QFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 39 R26P3H1MASAPISMLAMLFTLSGLRAQSVAQPEDQV alpha chainNVAEGNPLTVKCTYSVSGNPYLFWYVQYPN RGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMNRDDK IIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS 40R26P3H1 beta MSNQVLCCVVLCFLGANTVDGGITQSPKYL chainFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQ GLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSRAEGGEQ YFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 41 R35P3A4MTSIRAVFIFLWLQLDLVNGENVEQHPSTL alpha chainSVQEGDSAVIKCTYSDSASNYFPWYKQELG KRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASPTGGYNKL IFGAGTRLAVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 42R35P3A4 beta MSIGLLCCAALSLLWAGPVNAGVTQTPKFQ chainVLKTGQSMTLQCAQDMNHEYMSWYRQDPGM GLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSLGGASQE QYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 43 R37P1C9MKLVTSITVLLSLGIMGDAKTTQPNSMESN alpha chainEEEPVHLPCNHSTISGTDYIHWYRQLPSQG PEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILFNFNKFYFGSGTK LNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSS 44R37P1C9 beta MGPGLLHWMALCLLGTGHGDAMVIQNPRYQ chainVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQ APKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSSGETNEK LFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDF 45 R37P1H1MTRVSLLWAVVVSTCLESGMAQTVTQSQPE alpha chainMSVQEAETVTLSCTYDTSESNYYLFWYKQP PSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFGYSGGG ADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLSVIGFRILLLKVAGFNLLM TLRLWSS 46R37P1H1 beta MGPGLLCWALLCLLGAGLVDAGVTQSPTHL chainIKTRGQQVTLRCSPKSGHDTVSWYQQALGQ GPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSNEGQGWE AEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWW VNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDF 47 R42P3A9MKRILGALLGLLSAQVCCVRGIQVEQSPPD alpha chainLILQEGANSTLRCNFSDSVNNLQWFHQNPW GQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVHNFNKFYFGSG TKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQ NLSVIGFRILLLKVAGFNLLMTLRLWSS 48R42P3A9 beta MLSPDLPDSAWNTRLLCHVMLCLLGAVSVA chainAGVIQSPRHLIKEKRETATLKCYPIPRHDT VYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFC ASSLLGQGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFY PDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDSRG 49 R43P3F2MLTASLLRAVIASICVVSSMAQKVTQAQTE alpha chainISVVEKEDVTLDCVYETRDTTYYLFWYKQP PSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSNNNAG NMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS 50R43P3F2 beta MLSPDLPDSAWNTRLLCHVMLCLLGAVSVA chainAGVIQSPRHLIKEKRETATLKCYPIPRHDT VYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFC ASSPTGTSGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGF YPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKA TLYAVLVSALVLMAMVKRKDSRG 51 R43P3G5MEKNPLAAPLLILWFHLDCVSSILNVEQSP alpha chainQSLHVQEGDSTNFTCSFPSSNFYALHWYRW ETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALNRDDKI IFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 52R43P3G5 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYL beta chainVKRTGEKVFLECVQDMDHENMFWYRQDPGL GLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRLPSRTYE QYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 53 R59P2E7METLLGLLILWLQLQWWSSKQEVTQIPAAL alpha chainSVPEGENLVLNCSFTDSAIYNLQWFRQDPG KGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVNSDYKLSFG AGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLN FQNLSVIGFRILLLKVAGFNLLMTLRLWSS 54R59P2E7 beta MLSPDLPDSAWNTRLLCHVMLCLLGAVSVA chainAGVIQSPRHLIKEKRETATLKCYPIPRHDT VYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFC ASSLGLGTGDYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGF FPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKA TLYAVLVSALVLMAMVKRKDF 55 R11P3D3MEKNPLAAPLLILWFHLDCVSSILNVEQSP alpha chainQSLHVQEGDSTNFTCSFPSSNFYALHWYRW ETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDM RFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 56R11P3D3 beta MDSWTFCCVSLCILVAKHTDAGVIQSPRHE chainVTEMGQEVTLRCKPISGHNSLFWYRQTMMR GLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDT QYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 57 R16P1C10MKSLRVLLVILWLQLSWWSQQKEVEQNSGP alpha chainLSVPEGAIASLNCTYSDRGSQSFFWYRQYS GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEK LTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS 58R16P1C10 MGSRLLCWLLCLLGAGPVKAGVTQTPRYLI beta chainKTRGQQVTLSCSPISGHRSVSWYQQTPGQG LQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQ YFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 59 R16P1E8MMKSLRVLLVILWLQLSWVWSQQKEVEQDP alpha chainGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQ YSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNK LTFGGGTRVLVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS 60R16P1E8 beta MGTRLLCWAALCLLGAELTEAGVAQSPRYK chainIIEKRQSVAFWCNPISGHATLYWYQQILGQ GPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGE AFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDF 61 R17P1A9MKSLRVLLVILWLQLSWWWSQQKEVEQNSG alpha chainPLSVPEGAIASLNCTYSDRGSQSFFWYRQY SGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTAL IFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 62R17P1A9 beta MGFRLLCCVAFCLLGAGPVDSGVTQTPKHL chainITATGQRVTLRCSPRSGDLSVYWYQQSLDQ GLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWL GNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSW WWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG 63 R17P1D7MACPGFLWALVISTCLEFSMAQTVTQSQPE alpha chainMSVQEAETVTLSCTYDTSESDYYLFWYKQP PSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGG SEKLVFGKGTKLTVNPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSF ETDTNLNFQNLSVIGFRILLLKVAGFNLLM TLRLWSS 64R17P1D7 beta MTIRLLCYMGFYFLGAGLMEADIYQTPRYL chainVIGTGKKITLECSQTMGHDKMYWYQQDPGM ELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGT GELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWW WVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSE NDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG 65 R17P1G3IMSIYSNGDKEDGRFTAQLNKASQYVSLLI alpha chainRDSQPSDSATYLCAVGPSGTYKYIFGTGTR LKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSS 66 R17P1G3MGPQLLGYVVLCLLGAGPLEAQVTQNPRYL beta chain ITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEK RNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSE AEISHTQKATLVCLATGFYPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSS RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 67 R17P2B6 MKSLRVLLVILWLQLSWWWSQQKEVEQNSG alpha chainPLSVPEGAIASLNCTYSDRGSQSFFWYRQY SGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADG LTFGKGTHLIIQPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLR LWSS 68R17P2B6 beta MLSPDLPDSAWNTRLLCHVMLCLLGAVSVA chainAGVIQSPRHLIKEKRETATLKCYPIPRHDT VYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFC ASSLGRGGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFP DHVELSWWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDF 69 R11P3D3KEMEKNPLAAPLLILWFHLDCVSSILNVEQSP alpha chainQSLHVQEGDSTNFTCSFPSSNFYALHWYRK ETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDM RFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 70R11P3D3KE NNNVPIDDSGMPEDRFSAKMPNASFSTLKI beta chainQPSEPRDSAVYFCASSPGSTDTQYFGPGTR LTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWNGKEVHSGV STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP VTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKD SRG 71 R39P1C12TYLYWYKQEPGAGLQLLTYIFSNMDMKQDQ alpha chainRLTVLLNKKDKHLSLRIADTQTGDSAIYFC AEIDNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK DSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVK LVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 72 R39P1C12 MGPGLLCWALLCLLGAGLVDAGVTQSPTHL beta chainIKTRGQQVTLRCSPKSGHDTVSWYQQALGQ GPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSQLNTEAF FGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWWVNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDF 73 R39P1F5MKSLRVLLVILWLQLSWWSQQKEVEQNSGP alpha chainLSVPEGAIASLNCTYSDRGSQSFFWYRQYS GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNNARLMFGD GTQLWVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDM RSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNF QNLSVIGFRILLLKVAGFNLLMTLRLWSS 74R39P1F5 beta MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQ chainVTQMGQEVILRCVPISNHLYFYWYRQILGQ KVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGANE QYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 75 R40P1C2MACPGFLWALVISTCLEFSMAQTVTQSQPE alpha chainMSVQEAETVTLSCTYDTSESDYYLFWYKQP PSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYLNYQLI WGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLSVIGFRILLLKVAGFNLLMTLRLW SS 76R40P1C2 beta MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQ chainVTQMGQEVILRCVPISNHLYFYWYRQILGQ KVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEMTAVG QYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 77 R41P3E6MKSLRVLLVILWLQLSWWWSQQKEVEQNSG alpha chainPLSVPEGAIASLNCTYSDRGSQSFFWYRQY SGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAFSGYALNF GKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVL DMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNL NFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 78R41P3E6 beta MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQ chainVTQMGQEVILRCVPISNHLYFYWYRQILGQ KVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSQYTGEL FFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG 79 R43P3G4MKSLRVLLVILWLQLSWWSQQKEVEQNSGP alpha chainLSVPEGAIASLNCTYSDRGSQSFFWYRQYS GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNGGDMRFGA GTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDM RSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNF QNLSVIGFRILLLKVAGFNLLMTLRLWSS 80 R43P3G4MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQ beta chain VTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPD GSNFTLKIRSTKLEDSAMYFCASSGQGALEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSE AEISHTQKATLVCLATGFYPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSS RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 81 R44P3B3 MAMLLGASVLILWLQPDWNSQQKNDDQQVK alpha chainQNSPSLSVQEGRISILNCDYTNSMFDYFLW YKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGL YNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSD VYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVE KSFETDTNLNFQNLSVIGFRILLLKVAGFN LLMTLRLWSS82 R44P3B3 beta MGCRLLCCVVFCLLQAGPLDTAVSQTPKYL chainVTQMGNDKSIKCEQNLGHDTMYWYKQDSKK FLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSLGDRGYE QYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG 83 R44P3E7MKTFAGFSFLFLWLQLDCMSRGEDVEQSLF alpha chainLSVREGDSSVINCTYTDSSSTYLYWYKQEP GAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEINNNARLM FGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTV LDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLSVIGFRILLLKVAGFNLLMTLRLW SS 84R44P3E7 beta MLSPDLPDSAWNTRLLCHVMLCLLGAVSVA chainAGVIQSPRHLIKEKRETATLKCYPIPRHDT VYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFC ASSPPDQNTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPD HVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQ FYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLY AVLVSALVLMAMVKRKDSRG 85 R49P2B7MLLLLVPVLEVIFTLGGTRAQSVTQLGSHV alpha chainSVSEGALVLLRCNYSSSVPPYLFWYVQYPN QGLQLLLKYTTGATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVRIFGNEKL TFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 86R49P2B7 beta MGIRLLCRVAFCFLAVGLVDVKVTQSSRYL chainVKRTGEKVFLECVQDMDHENMFWYRQDPGL GLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLMGELTG ELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWW NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDSRG 87 R55P1G7MMKSLRVLLVILWLQLSWVWSQQKEVEQDP alpha chainGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQ YSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMMGDTGTA SKLTFGTGTRLQVTLDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFE TDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS 88R55P1G7 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYL beta chainVKRTGEKVFLECVQDMDHENMFWYRQDPGL GLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFGGYEQY FGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWWNGK EVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWT QDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMA MVKRKDSRG 89 R59P2A7VKPNIQNPDPAVYQLRDSKSSDKSVCLFTD alpha chainFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSV IGFRILLLKVAGFNLLMTLRLWSS 90 R59P2A7 betaMLCSLLALLLGTFFGVRSQTIHQWPATLVQ chain PVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQ FILSSKKLLLSDSGFYLCAWSGLVAEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEIS HTQKATLVCLATGFYPDHVELSWWWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGV LSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRG 91Variant MKSLRVLLVILWLQLSWWSQQKEVEQNSGP R4P3F9 alphaLSVPEGAIASLNCTYSDRRSQSFFWYRQYS chain GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQL TFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 92Variant MGFRLLCCVAFCLLGAGPVDSGVTQTPKHL R4P3F9 betaITATGQRVTLRCSPAMDHPYVYWYQQSLDQ chain GLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY TFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWWNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDF 93 Furin RXXR consensus 94 MSCVtgaaagaccccacctgtaggtttggcaagc promoter tagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagtt cagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggta agcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccct cagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgacc ctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgc tccccgagctcaataaaagagcccacaacc cctcact 95RD114TR MKLPTGMVILCSLIIVRAGFDDPRKAIALV QKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKISPSGGELQNCP CNTFQDSMHSSCYTEYRQCRRINKTYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSI NQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSL DARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQI IPPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGN NMAYTYLPQNWTRLCVQASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGI TAAFTTGATGLGVSVTQYTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLL TAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLASNPLWTGLQGFLPYLL PLLGPLLTLLLILTIGPCVFNRLVQFVKDRISVVQALVLTQQYHQLKPL

TABLE 4 TAA Peptide sequences SEQ ID NO: Amino Acid Sequence 99YLYDSETKNA 100 HLMDQPLSV 101 GLLKKINSV 102 FLVDGSSAL 103 FLFDGSANLV 104FLYKIIDEL 105 FILDSAETTTL 106 SVDVSPPKV 107 VADKIHSV 108 IVDDLTINL 109GLLEELVTV 110 TLDGAAVNQV 111 SVLEKEIYSI 112 LLDPKTIFL 113 YTFSGDVQL 114YLMDDFSSL 115 KVWSDVTPL 116 LLWGHPRVALA 117 KIWEELSVLEV 118 LLIPFTIFM119 FLIENLLAA 120 LLWGHPRVALA 121 FLLEREQLL 122 SLAETIFIV 123 TLLEGISRA124 KIQEILTQV 125 VIFEGEPMYL 126 SLFESLEYL 127 SLLNQPKAV 128 GLAEFQENV129 KLLAVIHEL 130 TLHDQVHLL 131 TLYNPERTITV 132 KLQEKIQEL 133 SVLEKEIYSI134 RVIDDSLVVGV 135 VLFGELPAL 136 GLVDIMVHL 137 FLNAIETAL 138 ALLQALMEL139 ALSSSQAEV 140 SLITGQDLLSV 141 QLIEKNWLL 142 LLDPKTIFL 143 RLHDENILL144 YTFSGDVQL 145 GLPSATTTV 146 GLLPSAESIKL 147 KTASINQNV 148 SLLQHLIGL149 YLMDDFSSL 150 LMYPYIYHV 151 KVWSDVTPL 152 LLWGHPRVALA 153 VLDGKVAVV154 GLLGKVTSV 155 KMISAIPTL 156 GLLETTGLLAT 157 TLNTLDINL 158 VIIKGLEEI159 YLEDGFAYV 160 KIWEELSVLEV 161 LLIPFTIFM 162 ISLDEVAVSL 163KISDFGLATV 164 KLIGNIHGNEV 165 ILLSVLHQL 166 LDSEALLTL 167 VLQENSSDYQSNL168 HLLGEGAFAQV 169 SLVENIHVL 170 YTFSGDVQL 171 SLSEKSPEV 172 AMFPDTIPRV173 FLIENLLAA 174 FTAEFLEKV 175 ALYGNVQQV 176 LFQSRIAGV 177 ILAEEPIYIRV178 FLLEREQLL 179 LLLPLELSLA 180 SLAETIFIV 181 AILNVDEKNQV 182 RLFEEVLGV183 YLDEVAFML 184 KLIDEDEPLFL 185 KLFEKSTGL 186 SLLEVNEASSV 187GVYDGREHTV 188 GLYPVTLVGV 189 ALLSSVAEA 190 TLLEGISRA 191 SLIEESEEL 192ALYVQAPTV 193 KLIYKDLVSV 194 ILQDGQFLV 195 SLLDYEVSI 196 LLGDSSFFL 197VIFEGEPMYL 198 ALSYILPYL 199 FLFVDPELV 200 SEWGSPHAAVP 201 ALSELERVL 202SLFESLEYL 203 KVLEYVIKV 204 VLLNEILEQV 205 SLLNQPKAV 206 KMSELQTYV 207ALLEQTGDMSL 208 VIIKGLEEITV 209 KQFEGTVEI 210 KLQEEIPVL 211 GLAEFQENV212 NVAEIVIHI 213 ALAGIVTNV 214 NLLIDDKGTIKL 215 VLMQDSRLYL 216KVLEHVVRV 217 LLWGNLPEI 218 SLMEKNQSL 219 KLLAVIHEL 220 ALGDKFLLRV 221FLMKNSDLYGA 222 KLIDHQGLYL 223 GPGIFPPPPPQP 224 ALNESLVEC 225 GLAALAVHL226 LLLEAVWHL 227 SIIEYLPTL 228 TLHDQVHLL 229 SLLMWITQC 230 FLLDKPQDLSI231 YLLDMPLWYL 232 GLLDCPIFL 233 VLIEYNFSI 234 TLYNPERTITV 235 AVPPPPSSV236 KLQEELNKV 237 KLMDPGSLPPL 238 ALIVSLPYL 239 FLLDGSANV 240 ALDPSGNQLI241 ILIKHLVKV 242 VLLDTILQL 243 HLIAEIHTA 244 SMNGGVFAV 245 MLAEKLLQA246 YMLDIFHEV 247 ALWLPTDSATV 248 GLASRILDA 249 ALSVLRLAL 250 SYVKVLHHL251 VYLPKIPSW 252 NYEDHFPLL 253 VYIAELEKI 254 VHFEDTGKTLLF 255 VLSPFILTL256 HLLEGSVGV

Example 2

Generation of WPRE Mutants

Wild-type WPRE sequences are used in lentiviral constructs to stabilizeand enhance transcription of genes. Due to some reports that concludethat a protein (X protein) within the WPRE can cause oncogenesis, the USFDA has recommended that lentiviral constructs used in clinical trialsfor gene and cellular therapy find alternatives to using wild type WPRE.We believe that fulfilling FDA requirements will enable our T cellproducts to be used in clinical trials and potentially avoid safetyconcerns with some aspects of lentiviral vector design.

Two separate WPRE mutation strategies were explored in an attempt todevelop WPRE mutants which do not express a functional X protein, whilemaintaining the post-transcriptional enhancement by WPRE on geneexpression.

One variant was developed in which both the promoter region of X proteinand the start codon of the X protein were mutated (SEQ ID NO: 4).

Another variant was developed in which the X protein promoter and fullputative sequence have been deleted along with mutating start codons ofany ORFs larger than 25aa within the WPRE (SEQ ID NO: 3).

Example 3

Lentiviral Constructs

A schematic of an expression cassette as used herein is provided in FIG.3 .

FIG. 4 provides a description of the cassettes used in the lentiviralconstructs used in the experiments detailed below to examine theefficacy of WPRE mutants.

The lentiviral vectors used herein contain several elements previouslyshown to enhance vector function, including a central polypurine tract(cPPT) for improved replication and nuclear import, a promoter from themurine stem cell virus (MSCV) (SEQ ID NO: 94), which has been shown tolessen vector silencing in some cell types, and the backbone has adeleted 3′-LTR self-inactivating (SIN) vector design that may haveimproved safety, sustained gene expression and anti-silencing properties(Yang et al. Gene Therapy (2008) 15, 1411-1423, the content of which isincorporated by reference in its entirety).

The lentiviral vectors used herein encode both a TCRα chain and a TCRβchain. In particular, the vectors used herein encode R4P3F9a and 13chains (SEQ ID NO: 25 and 26) and variants thereof. Vectors describedherein beginning with the abbreviation “R4” encode wild-type R4P3F9α andβ chains (SEQ ID NO: 25 and 26); vectors beginning with the abbreviation“R4-B4” encode a wild-type R4P3F9α chain (SEQ ID NO: 25) and a variantR4P3F9 β chain (SEQ ID NO: 92); and vectors beginning with theabbreviation “R4-A1B4” encode a variant R4P3F9α chain (SEQ ID NO: 91)and a variant R4P3F9 β chain (SEQ ID NO: 92) (FIG. 4 ).

For each TCRαβ dimer described above, four separate WPRE variations weretested. “Variant A” is the wild-type WPRE according to SEQ ID NO: 2(positive control); “variant B” contains no WPRE (negative control);“variant C” contains the mutant WPRE according to SEQ ID NO: 4 in whichthe X protein promoter and start codon have been mutated; and “variantD” contains the mutant WPRE according to SEQ ID NO: 3 in which startcodons located throughout the WPRE sequence have been mutated and the Xprotein promoter and ORF have been deleted.

Example 4

Effect of WPRE Mutation on Lentiviral Construct Efficacy in T Cells

T cells were obtained from donors on Day 0, activated on Day 1,transduced with the various lentiviral vectors described above inExample 3 on Day 2 and harvested on Day 6 for testing. TCR surfaceexpression was determined by flow cytometry and vector copy number wasdetermined by qPCR.

FIG. 5 shows HEK-293 T titers obtained following transduction withlentiviral constructs in accordance with some embodiments of the presentdisclosure. The titers obtained using lentiviral constructs containingmutant WPREs (LV-C & LV-D) were similar to those obtained usinglentiviral constructs containing wild-type (WT) WPRE (LV-A).

FIG. 6 shows expression of TCRs on the surface of CD8+ cells six daysafter transduction with R4-B4 lentiviral constructs in accordance withsome embodiments of the present disclosure. Expression was detected bytetramer using lentiviral titration in two separate donors: Donor #1 inpanel A and donor #2 in panel B. Log viral dilution factor is presentedalong the X-axis. Surprisingly, TCR expression was higher in CD8+ cellstransduced with lentiviral constructs containing mutant WPREs (variantsC & D) compared those transduced with lentiviral constructs containingeither WT WPRE (variant A) or no WPRE (variant B).

FIG. 7 shows expression of TCRs on the surface of CD8+ cells six daysafter transduction with R4-A1B4 lentiviral constructs in accordance withsome embodiments of the present disclosure. Expression was detected bytetramer using lentiviral titration in two separate donors: Donor #1 inpanel A and donor #2 in panel B. Log viral dilution factor is presentedalong the X-axis. Similar to the results from R4-B4 vectors shown inFIG. 6 , TCR expression was highest in CD8+ cells transduced withlentiviral constructs containing variant D (mutant WPRE according to SEQID NO: 3).

FIG. 8 shows expression of TCRs on the surface of CD8+ cells (A) or CD4+cells (B) six days after transduction with R4-B4 lentiviral constructsin accordance with some embodiments of the present disclosure.Expression was detected by tetramer using lentiviral titration. Logviral dilution factor is presented along the X-axis. These resultsfurther illustrate that TCR expression was higher in both CD8+ cells andCD4+ cells transduced with lentiviral constructs containing mutant WPREs(variants C & D) compared those transduced with lentiviral constructscontaining either WT WPRE (variant A) or no WPRE (variant B).

FIG. 9 shows expression of TCRs on the surface of CD8+ cells (A) or CD4+cells (B) six days after transduction with R4-A1B4 lentiviral constructsin accordance with some embodiments of the present disclosure.Expression was detected by tetramer using lentiviral titration. Logviral dilution factor is presented along the X-axis. Similar to theresults shown in FIGS. 6-8 , TCR expression was highest in CD8+ and CD4+cells transduced with lentiviral constructs containing variant D (mutantWPRE according to SEQ ID NO: 3). IMA203 is a lentiviral constructexpressing R11KE TCR and containing WT WPRE used as a negative control.

Fold expansion was not affected by WPRE mutations (FIG. 10 ). Cellviability was higher than 90% for all lentiviral constructs tested atoptimal MOI (data not shown).

WPRE mutants demonstrate comparable TCR tetramer surface expressionnormalized to vector copy number (FIG. 11 ) or normalized to viral titer(FIG. 12 ).

Similarly, FIG. 13 shows that WPRE mutants demonstrate comparable TCRtetramer surface expression as determined by flow cytometry. Panel Apresents CD4-CD8+/tetramer+ data. Panel B presents CD4+CD8-/tetramer+data.

FIG. 14 shows cytokine production of CD4+ or CD8+ T cells in thepresence of target-positive tumor cells. Panel A presents interferon-γ(IFN-γ) production in CD8+ T cells. Panel B presents IFN-γ production inCD4+ T cells. Panel C presents tumor necrosis factor-α (TNF-α)production in C8+ T cells. Panel D presents TNF-α production in CD4+ Tcells. MCF7=negative; SW982=460 CpC.

Example 5

γδ T Cell Manufacturing

To isolate γδ T cells, in an aspect, γδ T cells may be isolated from asubject or from a complex sample of a subject. In an aspect, a complexsample may be a peripheral blood sample, a cord blood sample, a tumor, astem cell precursor, a tumor biopsy, a tissue, a lymph, or fromepithelial sites of a subject directly contacting the external milieu orderived from stem precursor cells. γδ T cells may be directly isolatedfrom a complex sample of a subject, for example, by sorting γδ T cellsthat express one or more cell surface markers with flow cytometrytechniques. Wild-type γδ T cells may exhibit numerous antigenrecognition, antigen-presentation, co-stimulation, and adhesionmolecules that can be associated with a γδ T cells. One or more cellsurface markers, such as specific γδ TCRs, antigen recognition,antigen-presentation, ligands, adhesion molecules, or co-stimulatorymolecules may be used to isolate wild-type γδ T cells from a complexsample. Various molecules associated with or expressed by γδ T-cells maybe used to isolate γδ T cells from a complex sample, e.g., isolation ofmixed population of V61+, V02+, V03+ cells or any combination thereof.

For example, peripheral blood mononuclear cells can be collected from asubject, for example, with an apheresis machine, including theFicoll-Paque™ PLUS (GE Healthcare) system, or another suitabledevice/system. γδ T-cell(s), or a desired subpopulation of γδ T-cell(s),can be purified from the collected sample with, for example, with flowcytometry techniques. Cord blood cells can also be obtained from cordblood during the birth of a subject.

Positive and/or negative selection of cell surface markers expressed onthe collected γδ T cells can be used to directly isolate γδ T cells, ora population of γδ T cells expressing similar cell surface markers froma peripheral blood sample, a cord blood sample, a tumor, a tumor biopsy,a tissue, a lymph, or from an epithelial sample of a subject. Forinstance, γδ T cells can be isolated from a complex sample based onpositive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44,Kit, TCR α, TCR β, TCR α, TCR δ, NKG2D, CD70, CD27, CD30, CD16, CD337(NKp30), CD336 (NKp46), OX40, CD46, CCR7, and other suitable cellsurface markers.

FIG. 15 shows γδ T cell manufacturing according to an embodiment of thepresent disclosure. This process may include collecting or obtainingwhite blood cells or PBMC from leukapheresis products. Leukapheresis mayinclude collecting whole blood from a donor and separating thecomponents using an apheresis machine. An apheresis machine separatesout desired blood components and returns the rest to the donor'scirculation. For instance, white blood cells, plasma, and platelets canbe collected using apheresis equipment, and the red blood cells andneutrophils are returned to the donor's circulation. Commerciallyavailable leukapheresis products may be used in this process. Anotherway to obtain white blood cells is to obtain them from the buffy coat.To isolate the buffy coat, whole anticoagulated blood is obtained from adonor and centrifuged. After centrifugation, the blood is separated intoplasma, red blood cells, and buffy coat. The buffy coat is the layerlocated between the plasma and red blood cell layers. Leukapheresiscollections may result in higher purity and considerably increasedmononuclear cell content than that achieved by buffy coat collection.The mononuclear cell content possible with leukapheresis may typicallybe 20 times higher than that obtained from the buffy coat. In order toenrich for mononuclear cells, the use of a Ficoll gradient may be neededfor further separation.

To deplete αβ T cells from PBMC, TCR-expressing cells may be separatedfrom the PBMC by magnetic separation, e.g., using CliniMACS® magneticbeads coated with anti-αβ TCR antibodies, followed by cryopreservingTCR-T cells depleted PBMC. To manufacture “off-the-shelf” T-cellproducts, cryopreserved TCR-T cells depleted PBMC may be thawed andactivated in small/mid-scale, e.g., 24 to 4-6 well plates or T75/T175flasks, or in large scale, e.g., 50 ml-100 liter bags, in the presenceof aminobisphosphonate, e.g., zoledronate, and/orisopentenylpyrophosphate (IPP) and/or cytokines, e.g., interleukin 2(IL-2), interleukin 15 (IL-15), and/or interleukin 18 (IL-18), and/orother activators, e.g., Toll-like receptor 2 (TLR2) ligand, for 1-10days, e.g., 2-7 days.

FIG. 15 shows the activated T cells may be engineered by transducingwith a viral vector, such as lentiviral vector, expressing exogenousgenes of interest, such as αβ TCRs against specific cancer antigen andCD8, into isolated γδ T cells. Transduction may be carried out once ormultiple times to achieve stable transgene expression in small scale,e.g., 24 to 4-6 well plates, or mid/large scale for 1/2-5 days, e.g., 1day.

FIG. 16 further shows expansion of the transduced or engineered γδ Tcells may be carried out in the presence of cytokines, e.g., IL-2,IL-15, IL-18, and others, in small/mid-scale, e.g., flasks/G-Rex, or inlarge scale, e.g., 50 ml-100-liter bags, for 7-35 days, e.g., 7-28 days.The expanded transduced T cell products may then be cryopreserved as“off-the-shelf” T-cell products for infusion into patients.

Example 6

Comparison of γδ T Cells Transduced with Lentiviral Vectors (LV) withDifferent WPRE

FIG. 17 shows an example of γδ T cell manufacturing process, in which γδT cells transduced with LV expressing TCR (binding to SLLQHLIGL (SEQ IDNO: 148)/MHC complex) and CD8 having different WPRE were compared.Briefly, on Day 0, γδ T cells were activated in the presence ofzoledronate and cytokines and then transduced on Day 2 with a LVexpressing TCR and CD8 having the wild type (WT) WPRE (SEQ ID NO: 2)(A), no WPRE (B), WPREmut1 (SEQ ID NO: 4) (C), or WPREmut2 (SEQ ID NO:3) (D) at 3.75 μl, 7.50 μl, 15 μl, 30 μl, 60 μl, or 120 μl of LV per1×10⁶ cells. The LV titers of Batch #1 and Batch #2 are shown in Table5.

TABLE 5 Batch #1 Batch #2 LV Titer Titer WT WPRE 1.8 × 10⁸ IU/ml 7.56 ×10⁷ IU/ml WPREmut1 1.4 × 10⁸ IU/ml 6.47 × 10⁷ IU/ml WPREmut2 1.5 × 10⁸IU/ml 5.11 × 10⁷ IU/ml No WPRE 2.3 × 10⁸ IU/ml 5.85 × 10⁷ IU/ml

Table 5 shows that LV from Batch #1 have about 10-fold higher titersthan that from Batch #2. On Day 3, the transduced cells were expanded.On day 9, cells were counted and analyzed by FACS to measureTCR/CD8-expressing γδ T cells and copy number of integrated transgenes.

LV from Batch #1

Anti-Vβ8 antibody and anti-CD8α antibody were used to stain TCR+CD8α+ γδT cells in FACS analysis. FIG. 18A shows % Vβ8+CD8α+ γδ T cellsincreases with increasing amount of LV used in transduction. There is nosignificant difference in transduction efficiency between γδ T cellstransduced with LV having the wild type (WT) WPRE (A), no WPRE (B),WPREmut1 (C), and WPREmut2 (D). The non-transduced (NT) cells serve asnegative control. SLLQHLIGL (SEQ ID NO: 148)/MHC tetramer and anti-CD8αantibody were used to stain TCR+CD8α+ γδ T cells. FIG. 18B shows %tetramer+CD8α+ γδ T cells increases with increasing amount of LV used intransduction. There is no significant difference in transductionefficiency between γδ T cells transduced with LV having the wild type(WT) WPRE (A), no WPRE (B), WPREmut1 (C), and WPREmut2 (D). Thenon-transduced (NT) cells serve as negative control. Transductionefficiencies were then normalized to that of WT WPRE. There is nosignificant difference in normalized transduction efficiency between γδT cells transduced with LV having the wild type (WT) WPRE (A), no WPRE(B), WPREmut1 (C), and WPREmut2 (D) with respect to % Vβ8+CD8α+ γδ Tcells (FIG. 19A) and % tetramer+CD8α+ γδ T cells (FIG. 19B). Theseresults show transduction efficiencies are comparable among γδ T cellstransduced with LV having WT WPRE, WPREmut1, WPREmut2, and no WPRE.

FIG. 20 shows copy number of integrated transgenes in γδ T cellsgenerally increases with increasing amount of LV used in transduction.There is no significant difference in copy numbers of integratedtransgenes between γδ T cells transduced with LV having the wild type(WT) WPRE (A), no WPRE (B), WPREmut1 (C), and WPREmut2 (D). γδ T cellstransduced with LV with no WPRE (B) at 120 μl/1×10⁶ cells appears tohave slightly higher copy number of integrated transgenes than thattransduced with LV having different WPRE. Transduction efficiency/copynumber ratios were then determined. FIG. 21 shows that % Vβ8+CD8α+/copynumber ratios are comparable among γδ T cells transduced with LV havingWT WPRE, WPREmut1, WPREmut2, and no WPRE. Similarly, FIG. 22 shows that% tetramer+CD8α+/copy number ratios are comparable among γδ T cellstransduced with LV having WT WPRE, WPREmut1, WPREmut2, and no WPRE.

LV from Batch #2

As shown in Table 5, LV from Batch #1 have about 10-fold higher titersthan that from Batch #2. In general, transduction with LV from Batch #2resulted in lower transduction efficiency than that with LV from Batch#1 due to lower LV titers. FIG. 23 shows, at 120 μl LV/1×10⁶ cells, γδ Tcells obtained from Donors #4 and #5 transduced with LV having no WPREresulted in higher % Vβ8+CD8α+ γδ T cells (11.7% and 7.91%,respectively) than that transduced with WT WPRE (6.90% and 4.98%,respectively), WPREmut1 (6.01% and 3.71%, respectively), and WPREmut2(4.67% and 3.60%, respectively).

Table 6 shows the copy numbers of integrated transgenes of γδ T cellsobtained from Donors #4 and #5 transduced with LV having WT WPRE, noWPRE, WPREmut1, and WPREmut2. Overall, copy numbers of integratedtransgenes are lower than that of Batch #1 due to low LV titers.

TABLE 6 Volume LV contains μl/1 × 10⁶ cells Copy Number Donor #4 WT WPRE120 0.73 60 0.50 30 0.10 15 0.06 7.5 0.15 3.25 0.05 WPREmut1 120 1.11 600.78 30 0.42 15 0.18 7.5 0.09 3.25 0.05 WPREmut2 120 0.85 60 0.74 300.35 15 0.16 7.5 0.11 3.25 0.06 no WPRE 120 1.42 60 0.58 30 0.26 15 0.147.5 0.11 3.25 0.04 Non transduced N/A 0 Donor #5 WT WPRE 120 1.22 600.43 30 0.18 15 0.10 7.5 0.04 3.25 0.02 WPREmut1 120 0.46 60 0.33 300.34 15 0.13 7.5 0.06 3.25 0.04 WPREmut2 120 0.69 60 0.57 30 0.29 150.14 7.5 0.12 3.25 0.04 no WPRE 120 0.68 60 0.55 30 0.27 15 0.32 15 0.207.5 0.00 Non transduced N/A 0

FIG. 24 shows, at 120 μl LV/1×10⁶ cells, % tetramer+CD8α+/copy numberratios are comparable among γδ T cells obtained from Donors #4 and #5transduced with LV having WT WPRE (A), WPREmut1 (C), WPREmut2 (D), andno WPRE (B). FIG. 25 shows combined data, at 120 μl LV/1×10⁶ cells,obtained from Donors #3, #4, and #5. These combined results show %tetramer+CD8α+/copy number ratios are comparable among γδ T cellstransduced with LV having WT WPRE (A), WPREmut1 (C), WPREmut2 (D), andno WPRE (B). γδ T cells transduced with LV having no WPRE appear to haveless variation in % tetramer+CD8α+/copy number ratios than thattransduced with LV having different WPRE.

All references cited in this specification are herein incorporated byreference as though each reference was specifically and individuallyindicated to be incorporated by reference. The citation of any referenceis for its disclosure prior to the filing date and should not beconstrued as an admission that the present disclosure is not entitled toantedate such reference by virtue of prior invention.

It will be understood that each of the elements described above, or twoor more together may also find a useful application in other types ofmethods differing from the type described above. Without furtheranalysis, the foregoing will so fully reveal the gist of the presentdisclosure that others can, by applying current knowledge, readily adaptit for various applications without omitting features that, from thestandpoint of prior art, fairly constitute essential characteristics ofthe generic or specific aspects of this disclosure set forth in theappended claims. The foregoing embodiments are presented by way ofexample only; the scope of the present disclosure is to be limited onlyby the following claims.

What is claimed is:
 1. A vector comprising a mutant woodchuckpost-transcriptional regulatory element (WPRE) of SEQ ID NO: 1, whereinthe mutant WPRE comprises mutations in five or six start codons atpositions selected from the group consisting of 106-108, 152-154,245-247, 272-274, 283-285, and 362-364 of SEQ ID NO: 1, wherein themutant WPRE does not comprise an X protein promoter and wherein themutant WPRE does not comprise an X protein open reading frame (ORF). 2.The vector of claim 1, wherein the five or six start codons are mutatedat one, two, or all three positions within the start codon.
 3. Thevector of claim 1, wherein the five or six start codons are mutated fromATG to TTG.
 4. The vector of claim 1, further comprising a nucleotidesequence encoding a protein selected from the group of consisting ofenzymes, cytokines, chemokines, antibodies, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,immune co-stimulatory molecules, immunomodulatory molecules, a transdominant negative mutant of a target protein, a toxin, a conditionaltoxin, an antigenic peptide, an antigen receptor, a chimeric antigenreceptor, a T-cell receptor (TCR), a tumor suppressor protein, growthfactors, membrane proteins, pro- and anti-angiogenic proteins andpeptides, vasoactive proteins and peptides, antiviral proteins, andderivatives thereof.
 5. The vector of claim 1, comprising a firstnucleotide sequence S1 encoding a protein Z1 and a second nucleotidesequence S2 encoding a protein Z2, wherein Z1 and Z2 form a first dimer.6. The vector of claim 5, wherein the first dimer Z1Z2 is a T celldimeric signaling module, a TCR, an antibody, an antigen receptor, or achimeric antigen receptor.
 7. The vector of claim 5, wherein the firstdimer Z1Z2 is a TCR that binds to a target antigenic (TA) peptide, andwherein the target antigenic (TA) peptide is a viral peptide, abacterial peptide or a tumor associated antigen (TAA) antigenic peptide.8. The vector of claim 5, wherein the first dimer Z1Z2 is selected fromSEQ ID NO: 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and24, 25 and 26, 25 and 92, 91 and 92, 27 and 28, 29 and 30, 31 and 32, 33and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85and 86, 87 and 88, or 89 and
 90. 9. The vector of claim 5, furthercomprising a third nucleotide sequence S3 encoding a protein Y1 and afourth nucleotide sequence S4 encoding a protein Y2, wherein Y1 and Y2form a second dimer, wherein the first dimer Z1Z2 is structurallydifferent from the second dimer Y1Y2.
 10. The vector of claim 9, whereinthe second dimer Y1Y2 is a TCR co-receptor.
 11. The vector of claim 9,wherein the second dimer Y1Y2 is SEQ ID NO: 11 and
 12. 12. The vector ofclaim 1, further comprising a nucleotide sequence encoding a 2A peptideand a nucleotide sequence encoding a linker peptide.
 13. The vector ofclaim 1, further comprising a nucleotide sequence encoding a furincleavage site (SEQ ID NO: 10).
 14. The vector of claim 1, furthercomprising a promoter sequence selected from cytomegalovirus (CMV)promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein(MBP) promoter, glial fibrillary acidic protein (GFAP) promoter,modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer(MNDU3), Ubiqitin C promoter, EF-1 alpha promoter, or Murine Stem CellVirus (MSCV) promoter.
 15. The vector of claim 1, wherein the vector isa viral vector selected from adenoviruses, poxviruses, alphaviruses,arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses,herpesviruses, paramyxoviruses, or picornaviruses.
 16. The vector ofclaim 15, wherein the vector is pseudotyped with an envelope protein ofa virus selected from the native feline endogenous virus (RD114), achimeric version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), achimeric version of GALV (GALV-TR), amphotropic murine leukemia virus(MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G),fowl plague virus (FPV), Ebola virus (EboV), or baboon retroviralenvelope glycoprotein (BaEV), lymphocytic choriomeningitis virus (LCMV).17. The vector of claim 1, wherein the mutant WPRE comprises thenucleotide sequence at least 80% identity to SEQ ID NO:
 3. 18. Thevector of claim 1, further comprising a nucleotide sequence encoding anRNA selected from the group consisting of anti-sense RNA, smallinterfering RNA (siRNA), microRNA, shRNA, RNAi, and ribozyme.
 19. Thevector of claim 1, wherein the mutant WPRE comprises mutations in fivestart codons at positions selected from the group consisting of 106-108,152-154, 245-247, 272-274, 283-285, and 362-364 of SEQ ID NO:
 1. 20. Thevector of claim 1, wherein the mutant WPRE comprises mutations in startcodons at positions 106-108, 152-154, 245-247, 272-274, 283-285, and362-364 of SEQ ID NO: 1.